Modulation of SGLT2 expression

ABSTRACT

Compounds, compositions and methods are provided for modulating the expression of SGLT2. The compositions comprise oligonucleotides, targeted to nucleic acid encoding SGLT2. Methods of using these compounds for modulation of SGLT2 expression and for diagnosis and treatment of diseases and conditions associated with expression of SGLT2 are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority to U.S. provisionalpatent application Ser. No. 60/517,334 filed Nov. 3, 2003, and thebenefit of priority to U.S. application Ser. No. 10/946,498 filed Sep.21, 2004, each of which is incorporated herein by reference in itsentirety.

FIELD OF THE INVENTION

The present invention provides compositions and methods for modulatingthe expression of SGLT2. In particular, this invention relates toantisense compounds, particularly oligonucleotide compounds, which, insome embodiments, hybridize with nucleic acid molecules encoding SGLT2.Such compounds are shown herein to modulate the expression of SGLT2.

BACKGROUND OF THE INVENTION

A fundamental component of energy metabolism is glucose transport. Thetransport of glucose across cell membranes is essential to metabolicprocesses, including the maintenance of a relatively constant bloodglucose concentration and the delivery of glucose to peripheral tissuesfor storage and utilization. As cell membranes are essentiallyimpermeable to glucose, the movement of glucose across membranes must beaccomplished by protein transporters (Brown, J. Inherit. Metab. Dis.,2000, 23, 237-246).

Mediated glucose transport occurs in two forms, secondary activetransport and facilitated transport. In cells where glucose is rapidlymetabolized, the concentration gradient across the plasma membrane isused to drive facilitated transport, and an active mechanism is notrequired. Secondary active transport of glucose enables cells totransport glucose against a concentration gradient. This mechanisminvolves cotransport of glucose and sodium ions across the apicalsurface of the cells and the energy is provided by the sodium gradientmaintained by the sodium/potassium ATPase in the basolateral membrane.Efflux of glucose from the cells into the circulation is then mediatedby a facilitative transporter (Brown, J. Inherit. Metab. Dis., 2000, 23,237-246; Wright, Am. J. Physiol. Renal Physiol., 2001, 280, F10-18).

Secondary active transport of glucose operates in the mucosal cells ofthe intestine and the proximal tubular cells of the kidney and functionsto ensure efficient uptake of dietary glucose and minimal urinary loss.Plasma glucose is normally filtered in the kidney in the glomerulus andactively reabsorbed in the proximal tubule. Glucose is essentiallycompletely reabsorbed from the urine in the proximal tubule of thekidney through the action of the sodium-glucose cotransporters (SGLTs)located in the brush border membrane (BBM). Comparison of the glucosetransport properties of proximal tubule BBM vesicles prepared from theouter cortex and the outer medulla of rabbit kidney revealed thepresence of two distinct sodium-coupled D-glucose transport systems. Theouter cortex preparation exhibited a low-affinity/high-capacity activity(K_(m)=6 mM), whereas the outer medulla displayed ahigh-affinity/low-capacity activity (K_(m)=0.35 mM) (Turner and Moran,Am. J. Physiol. Endocrinol. Metab., 1982, 242, F406-414; Wright, Am. J.Physiol. Renal Physiol., 2001, 280, F10-18). Further characterization ofthe renal outer cortical BBM transport system revealed a glucose tosodium coupling ratio of 1:1, whereas the ratio is 2:1 in vesiclesisolated from the outer medullary tissue (Turner and Moran, J. Membr.Biol., 1982, 67, 73-80).

Isolation of nucleic acid molecules encoding SGLTs confirmed thepresence of multiple transport systems. A cDNA encoding human SGLT2(also known as solute carrier family 2, member 5, Na-dependent glucosecotransporter 2 or SLC2A5) was identified in a screen for sodiumcotransporter-like sequences in a cDNA library prepared from humankidney (Kanai et al., J. Clin. Invest., 1994, 93, 397-404; Wells et al.,Am. J. Physiol. Endocrinol. Metab., 1992, 263, F459-465). Human SGLT2localizes to chromosome 16p11.2 (Wells et al., Genomics, 1993, 17,787-789). Subsequent investigations of human SGLT2 revealed that hasfunctional properties characteristic of a low-affinity, sodium-dependentglucose cotransporter.

Studies of human SGLT2 injected into Xenopus oocytes demonstrated thatthis protein mediates sodium-dependent transport of D-glucose andα-methyl-D-glucopyranoside (α-MeGlc; a glucose analog) with a K_(m)value of 1.6 mM for α-MeGlc and a sodium to glucose coupling ratio of1:1 (Kanai et al., J. Clin. Invest., 1994, 93, 397-404; You et al., J.Biol. Chem., 1995, 270, 29365-29371). This transport activity wassuppressed by phlorizin, a plant glycoside that binds to the glucosesite but is not transported and thus inhibits SGLTs (You et al., J.Biol. Chem., 1995, 270, 29365-29371). These findings indicated thatSGLT2 is responsible for the low-affinity transport observed in BBMvesicle preparations from rabbit kidney outer cortex.

The tissue distribution of SGLT2 further suggested that thiscotransporter is the kidney low-affinity glucose transporter. Northernblotting revealed that human SGLT2 is primarily expressed in kidney, andin situ hybridization of a human SGLT2 probe to rat kidney tissuedemonstrated that SGLT2 is expressed in the proximal tubule S1 segmentsin the outer cortex (Kanai et al., J. Clin. Invest., 1994, 93, 397-404;Wells et al., Am. J. Physiol. Endocrinol. Metab., 1992, 263, F459-465).This localization pattern distinguishes SGLT2 from SGLT1, thehigh-affinity/low-capacity sodium/glucose transporter that is expressedin the proximal tubule S3 segments of the outer medulla, where it isappropriately positioned to reabsorb the remainder of filtered glucosenot reabsorbed by SGLT2 in the proximal tubule S1 segments.

Rat SGLT2, like human SGLT2, is strongly expressed in proximal S1segments and this expression is developmentally regulated, withexpression appearing on embryonic day 17, gradually increasing until day19 and subsequently decreasing between day 19 and birth. Interestingly,rat SGLT2 mRNA is 2.6 kb before birth and 2.2 kb after birth, suggestingthe presence of a different splice variant in embryonic kidney comparedto the adult (You et al., J. Biol. Chem., 1995, 270, 29365-29371).

The transport properties of rat SGLT2, i.e K_(m) of 3.0 mM and sodium toglucose coupling of 1:1, are also characteristic of a kidney corticallow-affinity transport system. Hybrid depletion studies in which ratkidney superficial cortex mRNA was mixed with an antisenseoligonucleotide corresponding to the 5′ portion of the rat SGLT2 codingregion completely suppressed the uptake of α-MeGlc in Xenopus oocytesinto which the mRNA/oligonucleotide mix was injected. An antisenseoligonucleotide targeted to SGLT1 had no effect on the uptake ofα-MeGlc. These data demonstrate that the α-MeGlc uptake was entirely dueto the expression of rat SGLT2 and support the proposal that SGLT2 isthe major kidney cortical low affinity glucose transporter (You et al.,J. Biol. Chem., 1995, 270, 29365-29371).

A second low-affinity SGLT, named SAAT-pSGLT2, was isolated from porcinekidney cells and was initially proposed to be the main low-affinityglucose transporter. However, further studies have revealed that themolecular characteristics of SAAT-pSGLT2 differ from those of SGLT2 andconsequently SAAT-pSGLT2 has been renamed SGLT3 (Kong et al., J. Biol.Chem., 1993, 268, 1509-1512; Mackenzie et al., J. Biol. Chem., 1996,271, 32678-32683; Mackenzie et al., J. Biol. Chem., 1994, 269,22488-22491; You et al., J. Biol. Chem., 1995, 270, 29365-29371).Whether SGLT3 contributes to glucose reabsorption in a physiologicallyrelevant manner is unclear.

The importance of SGLT2 function was demonstrated in hepatocyte nuclearfactor 1 α (HNF1α)-deficient animals, which are diabetic and also sufferfrom a renal Fanconi syndrome characterized by urinary glucose loss.HNF1α is a transcriptional activator expressed in liver, kidney,pancreas and intestine. The renal defect in these mice is due to an80-90% reduction in SGLT2 expression. Thus, HNF1α is one gene productthat controls SGLT2 expression, which is essential to proper glucosereabsorption in vivo (Pontoglio et al., EMBO Rep., 2000, 1, 359-365).

Reduction of SGLT2 mRNA was also observed upon exposure of mouse kidneycortical cells to cadmium, along with inhibition of sodium-dependentuptake of the glucose analog α-MeGlc. Interestingly, while both SGLT1and SGLT2 mRNA were decreased in mouse kidney cortical cells exposed tocadmium, SGLT3 mRNA was upregulated, suggesting that individual SGLTspecies are not regulated in a similar manner (Tabatabai et al.,Toxicol. Appl. Pharmacol., 2001, 177, 163-173). Changes in glucose orsodium filtrated rate also modulate the expression of sodium-glucosetransporter mRNA. Diabetic rats with glycosuria and rats fed a highsodium diet exhibited increased SGLT2 expression in the renal proximaltubule. The finding that SGLT1 levels in these rats were not altered tothe same extent as SGLT2 levels further supports the hypothesis that thecotransporters are differentially regulated (Vestri et al., J. Membr.Biol., 2001, 182, 105-112).

Although studies of SGLT function and localization in multiple mammalianspecies, including rat, mouse, pig, rabbit and dog, indicated that SGLT2is the low-affinity renal SGLT, the identity of the human SGLTresponsible for glucose reabsorption across the brush border of thehuman proximal tubule remained unclear. The lack of informationdescribing SGLT protein localization in renal brush border furtherhindered the identification of the human low-affinity SGLT. Moleculargenetic analysis of SGLT1 and SGLT2 indicated that a genetic alterationin the SGLT2 gene is a likely cause of renal glycosuria, a conditioncharacterized by elevated excretion of glucose in the urine (Hediger etal., Klin. Wochenschr., 1989, 67, 843-846). Direct evidence of SGLTfunction in the reabsorption of glucose came from analysis of the SGLT2gene in a patient with congenital isolated renal glucosuria. Sequenceanalysis revealed a homozygous nonsense mutation in exon 11 of the SGLT2gene leading to the formation of a truncated protein which is predictedto lack cotransport function (van den Heuvel et al., Hum. Genet., 2002,111, 544-547).

Whereas SGLT2 deficiency leads to inhibited reabsorption of glucose,SGLT2 elevation potentially allows for increased glucose uptake and isobserved in metastatic lesions of lung cancer. Quantitation of SGLT2gene expression revealed no significant difference between normal lungtissue and primary lung cancer. However, the metatstatic lesions of boththe liver and lymph node exhibited significantly higher expression ofSGLT2 (Ishikawa et al., Jpn. J. Cancer Res., 2001, 92, 874-879). Thisfinding is significant in light of evidence that different clinicaltumors show significantly increased glucose uptake in vivo compared tonormal tissue. Such a change in metabolism confers an advantage to tumorcells which allows them to survive and invade. Furthermore, glucoseuptake correlates with tumor aggressiveness and prognosis (Dang andSemenza, Trends Biochem. Sci., 1999, 24, 68-72).

Diabetes is a disorder characterized by hyperglycemia due to deficientinsulin action. Chronic hyperglycemia is a major risk factor fordiabetes-associated complications, including heart disease, retinopathy,nephropathy and neuropathy. As the kidneys play a major role in theregulation of plasma glucose levels, renal glucose transporters arebecoming attractive drug targets (Wright, Am. J. Physiol. RenalPhysiol., 2001, 280, F10-18). Synthetic agents that are derived fromphlorizin, a specific inhibitor of sodium/glucose transporters, havebeen designed and include T-1095, and its metabolically active formT-1095A (Tsujihara et al., J. Med. Chem., 1999, 42, 5311-5324).Phlorizin, T-1095 and T-1095A all inhibited sodium-dependent glucoseuptake in brush border membranes prepared from normal and diabetic ratkidney, rat small intestine, mouse kidney and dog kidney, as well as inXenopus oocytes injected with human SGLT mRNA (Oku et al., Diabetes,1999, 48, 1794-1800; Oku et al., Eur. J. Pharmacol., 2000, 391,183-192). These agents have been tested as antidiabetic compounds inlaboratory animals with genetic and streptozotocin-induced diabetes. Inthese models, administration of these compounds inhibited renal SGLTactivity, increased urinary glucose excretion and improved glucosetolerance, hyperglycemia and hypoinsulemia (Arakawa et al., Br. J.Pharmacol., 2001, 132, 578-586; Oku et al., Diabetes, 1999, 48,1794-1800; Oku et al., Eur. J. Pharmacol., 2000, 391, 183-192).Prolonged treatment of db/db mice with T-1095 yielded similar resultsand also almost completely suppressed the increase of urinary albuminand improved renal glomeruli pathology, indicating a beneficialinfluence on renal disfunction and a protective effect againstnephropathy, respectively (Arakawa et al., Br. J. Pharmacol., 2001, 132,578-586). Diabetic nephropathy is the most common cause of end-stagerenal disease that develops in many patients with diabetes. In Zuckerdiabetic fatty rats, long-term treatment with T-1095 lowered both fedand fasting glucose levels to near normal ranges. Also observed wererecovered hepatic glucose production and glucose utilization rateswithout a significant improvement in skeletal muscle glucose utilizationrate, indicating that hyperglycemia contributes to insulin resistance inhepatic and adipose tissue in this rat model of diabetes. These resultsfurther suggest that glucotoxicity, which results from long-termhyperglycemia, induces tissue-dependent insulin resistance in diabeticpatients (Nawano et al., Am. J. Physiol. Endocrinol. Metab., 2000, 278,E535-543).

Other SGLT2 inhibiting compounds are known in the art, such as thec-aryl glucosides disclosed in U.S. Pat. No. 6,414,126, which areinhibitors of sodium dependent glucose transporters found in theintestine and kidney and are proposed to treat diabetes, hyperglycemiaand related diseases when used alone or in combination with otherantidiabetic agents (Ellsworth et al., 2002).

The US pre-grant publication 20030055019 discloses isolated mutantproteins selected from a group which includes SGLT2, the correspondingnucleic acid molecules encoding said mutant proteins, isolated antisensederivatives of the nucleic acid sequences encoding said mutant proteins,as well as methods of delivering said antisense nucleic acid derivativesto treat or prevent hypertension, diabetes, insulin sensitivity,obesity, dyslipidemia and stroke. This application also discloses theantisense molecules may be DNA or RNA or a chimeric mixture,single-stranded or double-stranded or may comprise a ribozyme orcatalytic RNA (Shimkets, 2003).

The European Patent Applications EP 1 293 569 and EP 1 308 459 disclosea polynucleotide comprising a protein-coding region of the nucleotidesequence of any one of a group of sequences which includes a nucleicacid sequence encoding human SGLT2, an oligonucleotide comprising atleast 15 nucleotides complementary to the nucleotide sequence or to acomplementary strand thereof and an antisense polynucleotide against thedisclosed polynucleotide or a part thereof. These applications disclosethe use of said antisense polynucleotides for suppressing the expressionof a polypeptide of the invention and for gene therapy (Isogai et al.,2003, ; Isogai et al., 2003).

Although phlorizin and its derivatives are potent inhibitors ofsodium-glucose cotransporters, these agents do not specifically inhibita single species of SGLT, thus all SGLTs in all tissues are affected.Thus, there remains a need for therapeutic compounds that targetsspecific SGLT species. Antisense technology is an effective means forreducing the expression of specific gene products and may thereforeprove to be uniquely useful in a number of therapeutic, diagnostic andresearch applications for the modulation of SGLT2 expression.

The present invention provides compounds and methods for modulatingSGLT2 expression.

SUMMARY OF THE INVENTION

The present invention is directed to oligomeric compounds, especiallynucleic acid and nucleic acid-like oligomers, such as antisensecompounds, which are targeted to a nucleic acid encoding SGLT2, andwhich modulate the expression of SGLT2. Pharmaceutical and othercompositions comprising the compounds of the invention are alsoprovided. Further provided are methods of screening for modulators ofSGLT2 and methods of modulating the expression of SGLT2 in cells,tissues or animals comprising contacting the cells, tissues or animalswith one or more of the compounds or compositions of the invention.Further provided are diagnostic methods for identifying a disease stateby identifying the presence of SGLT2 in a sample using one or more ofthe compounds of the invention. Methods of treating an animal,particularly a human, suspected of having or being prone to a disease orcondition associated with expression of SGLT2 are also set forth herein.Such methods comprise administering a therapeutically orprophylactically effective amount of one or more of the compounds orcompositions of the invention to the person, who may be in need oftreatment.

Also provided are methods of enhancing inhibition of expression ofpreselected cellular RNA targets in kidney cells and kidney tissue usingcompounds, such as antisense compounds, of the invention. Furtherprovided are methods of preventing or delaying the onset of a disease orcondition in an animal, wherein the disease or condition is associatedwith expression of a preselected cellular RNA target expressed in thekidney, particularly SGLT2. Methods of lowering blood glucose levels inan animal and methods of delaying or preventing the onset of type 2diabetes also are set forth herein. Such methods comprise administeringa therapeutically or prophylactically effective amount of one or more ofthe compounds of the invention to the animal, which may be in need oftreatment. Provided herein are methods of enhancing inhibition ofexpression of SGLT2 in kidney cells or kidney tissues, comprisingcontacting the cells or tissues with one or more of the compounds of theinvention, such as antisense compounds.

DETAILED DESCRIPTION OF THE INVENTION

The present invention employs oligomeric compounds, preferablyoligonucleotides and similar species, such as antisense compounds, foruse in modulating the function or effect of nucleic acid moleculesencoding SGLT2. This is accomplished by providing oligomeric compounds,such as oligonucleotides, which specifically hybridize with one or morenucleic acid molecules encoding SGLT2.

In one embodiment, the oligomeric compounds of the invention arechimeric oligonucleotides (“gapmers”), composed of a central “gap”region consisting of 2′-deoxynucleotides, which is flanked on both sides(5′ and 3′ directions) by “wings” composed of 2′-methoxyethyl (2′-MOE)nucleotides. In some embodiments, the internucleoside (backbone)linkages are phosphorothioate (P═S) throughout the oligonucleotide. Insome embodiments, one or more cytidine residues are 5-methylcytidines.

In another embodiment, the oligomeric compounds of the invention arechimeric oligonucleotides having mixed phosphorothioate andphosphodiester backbones, referred to herein as “mixed backbonecompounds.” The mixed backbone compounds of the invention can have acentral “gap” region consisting of at least 5 contiguous 2′-deoxynucleosides flanked by two “wing” regions consisting of at least one2′-O-methoxyethyl nucleoside in each region. The internucleosidelinkages of the mixed backbone compounds can be phosphorothioatelinkages in the central “gap” region and phosphodiester linkages in thetwo “wing” regions. In another embodiment, mixed backbone compounds havephosphodiester linkages in the “wing” regions except for onephosphodiester linkage at one or both of the extreme 5′ and 3′ ends ofthe oligonucleotide.

It is shown herein that mixed backbone compounds are efficientlydelivered to the kidney and treatment with the mixed backbone compoundsresults in efficient modulation of target gene expression in the kidneywithout liver or kidney toxicity. It is further shown herein thattreatment with mixed backbone compounds in animal models of type 2diabetes reduces blood glucose levels in diabetic animals.

As used herein, the terms “target nucleic acid” and “nucleic acidmolecule encoding SGLT2” have been used for convenience to encompass DNAencoding SGLT2, RNA (including pre-mRNA and mRNA or portions thereof)transcribed from such DNA, and also cDNA derived from such RNA. Thehybridization of a compound of this invention with its target nucleicacid is generally referred to as “antisense.” Consequently, onemechanism believed to be included in the practice of some embodiments ofthe invention is referred to herein as “antisense inhibition.” Suchantisense inhibition is typically based upon hydrogen bonding-basedhybridization of oligonucleotide strands or segments such that at leastone strand or segment is cleaved, degraded, or otherwise renderedinoperable. In this regard, specific nucleic acid molecules and theirfunctions can be targeted for such antisense inhibition.

The functions of DNA to be interfered with can include replication andtranscription. Replication and transcription, for example, can be froman endogenous cellular template, a vector, a plasmid construct orotherwise. The functions of RNA to be interfered with can includefunctions such as, for example, translocation of the RNA to a site ofprotein translation, translocation of the RNA to sites within the cellwhich are distant from the site of RNA synthesis, translation of proteinfrom the RNA, splicing of the RNA to yield one or more RNA species, andcatalytic activity or complex formation involving the RNA which may beengaged in or facilitated by the RNA. One result of such interferencewith target nucleic acid function is modulation of the expression ofSGLT2. In the context of the present invention, “modulation” and“modulation of expression” mean either an increase (stimulation) or adecrease (inhibition) in the amount or levels of a nucleic acid moleculeencoding the gene, e.g., DNA or RNA. Inhibition is often the desiredform of modulation of expression and mRNA is often a desired targetnucleic acid.

In the context of this invention, “hybridization” means the pairing ofcomplementary strands of oligomeric compounds. In the present invention,one mechanism of pairing involves hydrogen bonding, which may beWatson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, betweencomplementary nucleoside or nucleotide bases (nucleobases) of thestrands of oligomeric compounds. For example, adenine and thymine arecomplementary nucleobases which pair through the formation of hydrogenbonds. Hybridization can occur under varying circumstances.

An oligomeric compound, such as an antisense compound, is specificallyhybridizable when binding of the compound to the target nucleic acidinterferes with the normal function of the target nucleic acid to causea loss of activity, and there is a sufficient degree of complementarityto avoid non-specific binding of the antisense compound to non-targetnucleic acid sequences under conditions in which specific binding isdesired, i.e., under physiological conditions in the case of in vivoassays or therapeutic treatment, and under conditions in which assaysare performed in the case of in vitro assays.

In the present invention the phrase “stringent hybridization conditions”or “stringent conditions” refers to conditions under which a compound ofthe invention will hybridize to its target sequence, but to a minimalnumber of other sequences. Stringent conditions are sequence-dependentand will be different in different circumstances and in the context ofthis invention, “stringent conditions” under which oligomeric compoundshybridize to a target sequence are determined by the nature andcomposition of the oligomeric compounds and the assays in which they arebeing investigated.

“Complementary,” as used herein, refers to the capacity for precisepairing between two nucleobases of an oligomeric compound. For example,if a nucleobase at a certain position of an oligonucleotide (anoligomeric compound), is capable of hydrogen bonding with a nucleobaseat a certain position of a target nucleic acid, said target nucleic acidbeing a DNA, RNA, or oligonucleotide molecule, then the position ofhydrogen bonding between the oligonucleotide and the target nucleic acidis considered to be a complementary position. The oligonucleotide andthe further DNA, RNA, or oligonucleotide molecule are complementary toeach other when a sufficient number of complementary positions in eachmolecule are occupied by nucleobases which can hydrogen bond with eachother. Thus, “specifically hybridizable” and “complementary” are termswhich are used to indicate a sufficient degree of precise pairing orcomplementarity over a sufficient number of nucleobases such that stableand specific binding occurs between the oligonucleotide and a targetnucleic acid.

It is understood in the art that the sequence of an oligomeric compoundneed not be 100% complementary to that of its target nucleic acid to bespecifically hybridizable. Moreover, an oligomeric compound mayhybridize over one or more segments such that intervening or adjacentsegments are not involved in the hybridization event (e.g., a loopstructure or hairpin structure). The antisense compounds of the presentinvention can comprise at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, or at least 99% sequencecomplementarity to the target region within the target nucleic acidsequence to which they are targeted. For example, an antisense compoundin which 18 of 20 nucleobases of the antisense compound arecomplementary to a target region, and would therefore specificallyhybridize, would represent 90 percent complementarity. In this example,the remaining noncomplementary nucleobases may be clustered orinterspersed with complementary nucleobases and need not be contiguousto each other or to complementary nucleobases. As such, an antisensecompound which is 18 nucleobases in length having 4 (four)noncomplementary nucleobases which are flanked by two regions ofcomplete complementarity with the target nucleic acid would have 77.8%overall complementarity with the target nucleic acid and would thus fallwithin the scope of the present invention. Percent complementarity of anantisense compound with a region of a target nucleic acid can bedetermined routinely using BLAST programs (basic local alignment searchtools) and PowerBLAST programs known in the art (Altschul et al., J.Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7,649-656).

Percent homology, sequence identity or complementarity, can bedetermined by, for example, the Gap program (Wisconsin Sequence AnalysisPackage, Version 8 for Unix, Genetics Computer Group, UniversityResearch Park, Madison Wis.), using default settings, which uses thealgorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482-489). Insome embodiments, homology, sequence identity or complementarity,between the oligomeric and target is from about 50% to about 60%, fromabout 60% to about 70%, from about 70% to about 80%, from about 80% toabout 90%, about 90%, about 92%, about 94%, about 95%, about 96%, about97%, about 98%, about 99%, or about 100%. As used herein, the term“about” means ±5% of the value modified.

According to the present invention, oligomeric compounds, such asantisense compounds, include antisense oligomeric compounds, antisenseoligonucleotides, ribozymes, external guide sequence (EGS)oligonucleotides, alternate splicers, and other oligomeric compoundswhich hybridize to at least a portion of the target nucleic acid. Assuch, these compounds may be introduced in the form of single-stranded,double-stranded, circular or hairpin oligomeric compounds and maycontain structural elements such as internal or terminal bulges orloops. Once introduced to a system, the compounds of the invention mayelicit the action of one or more enzymes or structural proteins toeffect modification of the target nucleic acid.

One non-limiting example of such an enzyme is RNAse H, a cellularendonuclease which cleaves the RNA strand of an RNA:DNA duplex. It isknown in the art that single-stranded antisense compounds which are“DNA-like” elicit RNAse H. Activation of RNase H, therefore, results incleavage of the RNA target, thereby greatly enhancing the efficiency ofoligonucleotide-mediated inhibition of gene expression. Similar roleshave been postulated for other ribonucleases such as those in the RNaseIII and ribonuclease L family of enzymes.

While one form of antisense compound is a single-stranded antisenseoligonucleotide, in many species the introduction of double-strandedstructures, such as double-stranded RNA (dsRNA) molecules, has beenshown to induce potent and specific antisense-mediated reduction of thefunction of a gene or its associated gene products. This phenomenonoccurs in both plants and animals and is believed to have anevolutionary connection to viral defense and transposon silencing.

The first evidence that dsRNA could lead to gene silencing in animalscame in 1995 from work in the nematode, Caenorhabditis elegans (Guo andKempheus, Cell, 1995, 81, 611-620). Montgomery et al. have shown thatthe primary interference effects of dsRNA are posttranscriptional(Montgomery et al., Proc. Natl. Acad. Sci. USA, 1998, 95, 15502-15507).The posttranscriptional antisense mechanism defined in Caenorhabditiselegans resulting from exposure to double-stranded RNA (dsRNA) has sincebeen designated RNA interference (RNAi). This term has been generalizedto mean antisense-mediated gene silencing involving the introduction ofdsRNA leading to the sequence-specific reduction of endogenous targetedmRNA levels (Fire et al., Nature, 1998, 391, 806-811). Recently, it hasbeen shown that it is, in fact, the single-stranded RNA oligomers ofantisense polarity of the dsRNAs which are the potent inducers of RNAi(Tijsterman et al., Science, 2002, 295, 694-697).

The oligomeric compounds of the present invention also include modifiedcompounds in which a different base is present at one or more of thenucleotide positions in the compound. For example, if the firstnucleotide is an adenosine, modified compounds may be produced whichcontain thymidine, guanosine or cytidine at this position. This may bedone at any of the positions of the antisense compound. These compoundsare then tested using the methods described herein to determine theirability to inhibit expression of SGLT2 mRNA.

In the context of this invention, the term “oligomeric compound” refersto a polymer or oligomer comprising a plurality of monomeric units. Inthe context of this invention, the term “oligonucleotide” refers to anoligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid(DNA) or mimetics, chimeras, analogs and homologs thereof. This termincludes oligonucleotides composed of naturally occurring nucleobases,sugars and covalent internucleoside (backbone) linkages as well asoligonucleotides having non-naturally occurring portions which functionsimilarly. Such modified or substituted oligonucleotides are oftendesired over native forms because of desirable properties such as, forexample, enhanced cellular uptake, enhanced affinity for a targetnucleic acid and increased stability in the presence of nucleases.

While oligonucleotides are one form of the antisense compounds of thisinvention, the present invention comprehends other families of antisensecompounds as well, including but not limited to oligonucleotide analogsand mimetics such as those described herein.

The antisense compounds in accordance with this invention can comprisefrom about 8 to about 80 nucleobases (i.e. from about 8 to about 80linked nucleosides). One of ordinary skill in the art will appreciatethat the invention embodies compounds of 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 nucleobases in length, orany range therewithin.

In one embodiment, the antisense compounds of the invention are 10 to 50nucleobases in length. One having ordinary skill in the art willappreciate that this embodies compounds of 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50nucleobases in length, or any range therewithin.

In another embodiment, the antisense compounds of the invention are 13to 30 nucleobases in length. One having ordinary skill in the art willappreciate that this embodies compounds of 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleobases in length, orany range therewithin.

In another embodiment, the antisense compounds of the invention are 15to 25 nucleobases in length. One having ordinary skill in the art willappreciate that this embodies compounds of 15, 16, 17, 18, 19, 20, 21,22, 23, 24 or 25 nucleobases in length, or any range therewithin.

In another embodiment, the antisense compounds of the invention are 18to 22 nucleobases in length. One having ordinary skill in the art willappreciate that this embodies compounds of 18, 19, 20, 21 or 22nucleobases in length, or any range therewithin.

Particularly suitable compounds are oligonucleotides from about 10 toabout 50 nucleobases, from about 13 to about 30 nucleobases, from about15 to about 25, and from about 18 to about 22 nucleobases.

Antisense compounds 8 to 80 nucleobases in length comprising a stretchof at least eight (8) consecutive nucleobases selected from within theillustrative antisense compounds are considered to be suitable antisensecompounds as well.

Exemplary antisense compounds include oligonucleotide sequences thatcomprise at least the 8 consecutive nucleobases from the 5′-terminus ofone of the illustrative antisense compounds (the remaining nucleobasesbeing a consecutive stretch of the same oligonucleotide beginningimmediately upstream of the 5′-terminus of the antisense compound whichis specifically hybridizable to the target nucleic acid and continuinguntil the oligonucleotide contains about 8 to about 80 nucleobases).Similarly suitable antisense compounds are represented byoligonucleotide sequences that comprise at least the 8 consecutivenucleobases from the 3′-terminus of one of the illustrative antisensecompounds (the remaining nucleobases being a consecutive stretch of thesame oligonucleotide beginning immediately downstream of the 3′-terminusof the antisense compound which is specifically hybridizable to thetarget nucleic acid and continuing until the oligonucleotide containsabout 8 to about 80 nucleobases). It is also understood that antisensecompounds may be represented by oligonucleotide sequences that compriseat least 8 consecutive nucleobases from an internal portion of thesequence of an illustrative antisense compound, and may extend in eitheror both directions until the oligonucleotide contains about 8 to about80 nucleobases.

One having skill in the art armed with the antisense compoundsillustrated herein will be able, without undue experimentation, toidentify additional antisense compounds.

“Targeting” an antisense compound to a particular nucleic acid molecule,in the context of this invention, can be a multistep process. Theprocess usually begins with the identification of a target nucleic acidwhose function is to be modulated. This target nucleic acid may be, forexample, a cellular gene (or mRNA transcribed from the gene) whoseexpression is associated with a particular disorder or disease state, ora nucleic acid molecule from an infectious agent. In the presentinvention, the target nucleic acid encodes SGLT2.

The targeting process usually also includes determination of at leastone target region, segment, or site within the target nucleic acid forthe antisense interaction to occur such that the desired effect, e.g.,modulation of expression, will result. Within the context of the presentinvention, the term “region” is defined as a portion of the targetnucleic acid having at least one identifiable structure, function, orcharacteristic. Within regions of target nucleic acids are segments.“Segments” are defined as smaller or sub-portions of regions within atarget nucleic acid. “Sites,” as used in the present invention, aredefined as positions within a target nucleic acid.

Since, as is known in the art, the translation initiation codon istypically 5′-AUG (in transcribed mRNA molecules; 5′-ATG in thecorresponding DNA molecule), the translation initiation codon is alsoreferred to as the “AUG codon,” the “start codon” or the “AUG startcodon.” A minority of genes have a translation initiation codon havingthe RNA sequence 5′-GUG, 5′-UUG or 5′-CUG, and 5′-AUA, 5′-ACG and 5′-CUGhave been shown to function in vivo. Thus, the terms “translationinitiation codon” and “start codon” can encompass many codon sequences,even though the initiator amino acid in each instance is typicallymethionine (in eukaryotes) or formylmethionine (in prokaryotes). It isalso known in the art that eukaryotic and prokaryotic genes may have twoor more alternative start codons, any one of which may be preferentiallyutilized for translation initiation in a particular cell type or tissue,or under a particular set of conditions. In the context of theinvention, “start codon” and “translation initiation codon” refer to thecodon or codons that are used in vivo to initiate translation of an mRNAtranscribed from a gene encoding SGLT2, regardless of the sequence(s) ofsuch codons. It is also known in the art that a translation terminationcodon (or “stop codon”) of a gene may have one of three sequences, i.e.,5′-UAA, 5′-UAG and 5′-UGA (the corresponding DNA sequences are 5′-TAA,5′-TAG and 5′-TGA, respectively).

The terms “start codon region” and “translation initiation codon region”refer to a portion of such an mRNA or gene that encompasses from about25 to about 50 contiguous nucleotides in either direction (i.e., 5′ or3′) from a translation initiation codon. Similarly, the terms “stopcodon region” and “translation termination codon region” refer to aportion of such an mRNA or gene that encompasses from about 25 to about50 contiguous nucleotides in either direction (i.e., 5′ or 3′) from atranslation termination codon. Consequently, the “start codon region”(or “translation initiation codon region”) and the “stop codon region”(or “translation termination codon region”) are all regions which may betargeted effectively with the antisense compounds of the presentinvention.

The open reading frame (ORF) or “coding region,” which is known in theart to refer to the region between the translation initiation codon andthe translation termination codon, is also a region which may betargeted effectively. Within the context of the present invention, asuitable region is the intragenic region encompassing the translationinitiation or termination codon of the open reading frame (ORF) of agene.

Other target regions include the 5′ untranslated region (5′UTR), knownin the art to refer to the portion of an mRNA in the 5′ direction fromthe translation initiation codon, and thus including nucleotides betweenthe 5′ cap site and the translation initiation codon of an mRNA (orcorresponding nucleotides on the gene), and the 3′ untranslated region(3′UTR), known in the art to refer to the portion of an mRNA in the 3′direction from the translation termination codon, and thus includingnucleotides between the translation termination codon and 3′ end of anmRNA (or corresponding nucleotides on the gene). The 5′ cap site of anmRNA comprises an N7-methylated guanosine residue joined to the 5′-mostresidue of the mRNA via a 5′-5′ triphosphate linkage. The 5′ cap regionof an mRNA is considered to include the 5′ cap structure itself as wellas the first 50 nucleotides adjacent to the cap site. It is alsosuitable to target the 5′ cap region.

Although some eukaryotic mRNA transcripts are directly translated, manycontain one or more regions, known as “introns,” which are excised froma transcript before it is translated. The remaining (and thereforetranslated) regions are known as “exons” and are spliced together toform a continuous mRNA sequence. Targeting splice sites, i.e.,intron-exon junctions or exon-intron junctions, may also be particularlyuseful in situations where aberrant splicing is implicated in disease,or where an overproduction of a particular splice product is implicatedin disease. Aberrant fusion junctions due to rearrangements or deletionsare also suitable target sites. mRNA transcripts produced via theprocess of splicing of two (or more) mRNAs from different gene sourcesare known as “fusion transcripts.” It is also known that introns can beeffectively targeted using antisense compounds targeted to, for example,DNA or pre-mRNA.

It is also known in the art that alternative RNA transcripts can beproduced from the same genomic region of DNA. These alternativetranscripts are generally known as “variants.” More specifically,“pre-mRNA variants” are transcripts produced from the same genomic DNAthat differ from other transcripts produced from the same genomic DNA ineither their start or stop position and contain both intronic and exonicsequence.

Upon excision of one or more exon or intron regions, or portions thereofduring splicing, pre-mRNA variants produce smaller “mRNA variants.”Consequently, mRNA variants are processed pre-mRNA variants and eachunique pre-mRNA variant must always produce a unique mRNA variant as aresult of splicing. These mRNA variants are also known as “alternativesplice variants.” If no splicing of the pre-mRNA variant occurs then thepre-mRNA variant is identical to the mRNA variant.

It is also known in the art that variants can be produced through theuse of alternative signals to start or stop transcription and thatpre-mRNAs and mRNAs can possess more that one start codon or stop codon.Variants that originate from a pre-mRNA or mRNA that use alternativestart codons are known as “alternative start variants” of that pre-mRNAor mRNA. Those transcripts that use an alternative stop codon are knownas “alternative stop variants” of that pre-mRNA or mRNA. One specifictype of alternative stop variant is the “polyA variant” in which themultiple transcripts produced result from the alternative selection ofone of the “polyA stop signals” by the transcription machinery, therebyproducing transcripts that terminate at unique polyA sites. Within thecontext of the invention, the types of variants described herein arealso suitable target nucleic acids.

The locations on the target nucleic acid to which the antisensecompounds hybridize are hereinbelow referred to as “suitable targetsegments.” As used herein the term “suitable target segment” is definedas at least an 8-nucleobase portion of a target region to which anactive antisense compound is targeted. While not wishing to be bound bytheory, it is presently believed that these target segments representportions of the target nucleic acid which are accessible forhybridization.

While the specific sequences of certain preferred target segments areset forth herein, one of skill in the art will recognize that theseserve to illustrate and describe particular embodiments within the scopeof the present invention. Additional target segments may be identifiedby one having ordinary skill.

Target segments 8-80 nucleobases in length comprising a stretch of atleast eight (8) consecutive nucleobases selected from within theillustrative target segments are considered to be suitable for targetingas well.

Target segments can include DNA or RNA sequences that comprise at leastthe 8 consecutive nucleobases from the 5′-terminus of one of theillustrative target segments (the remaining nucleobases being aconsecutive stretch of the same DNA or RNA beginning immediatelyupstream of the 5′-terminus of the target segment and continuing untilthe DNA or RNA contains about 8 to about 80 nucleobases). Targetsegments are also represented by DNA or RNA sequences that comprise atleast the 8 consecutive nucleobases from the 3′-terminus of one of theillustrative target segments (the remaining nucleobases being aconsecutive stretch of the same DNA or RNA beginning immediatelydownstream of the 3′-terminus of the target segment and continuing untilthe DNA or RNA contains about 8 to about 80 nucleobases). It is alsounderstood that antisense target segments may be represented by DNA orRNA sequences that comprise at least 8 consecutive nucleobases from aninternal portion of the sequence of an illustrative target segment, andmay extend in either or both directions until the oligonucleotidecontains about 8 to about 80 nucleobases. One having skill in the artarmed with the target segments illustrated herein will be able, withoutundue experimentation, to identify further target segments.

Once one or more target regions, segments or sites have been identified,antisense compounds are chosen which are sufficiently complementary tothe target, i.e., hybridize sufficiently well and with sufficientspecificity, to give the desired effect.

The oligomeric antisense compounds may also be targeted to regions ofthe target nucleobase sequence (e.g., such as those disclosed in Example16) comprising nucleobases 1-80, 81-160, 161-240, 241-320, 321-400,401-480, 481-560, 561-640, 641-720, 721-800, 801-880, 881-960, 961-1040,1041-1120, 1121-1200, 1201-1280, 1281-1360, 1361-1440, 1441-1520,1521-1600, 1601-1680, 1681-1760, 1761-1840, 1841-1920, 1921-2000,2001-2080, 2081-2160, 2161-2240, 2241-2273, or any combination thereof.

In a further embodiment, the “suitable target segments” identifiedherein may be employed in a screen for additional compounds thatmodulate the expression of SGLT2. “Modulators” are those compounds thatdecrease or increase the expression of a nucleic acid molecule encodingSGLT2 and which comprise at least an 8-nucleobase portion which iscomplementary to a target segment. The screening method comprises thesteps of contacting a target segment of a nucleic acid molecule encodingSGLT2 with one or more candidate modulators, and selecting for one ormore candidate modulators which decrease or increase the expression of anucleic acid molecule encoding SGLT2. Once it is shown that thecandidate modulator or modulators are capable of modulating (e.g. eitherdecreasing or increasing) the expression of a nucleic acid moleculeencoding SGLT2, the modulator may then be employed in furtherinvestigative studies of the function of SGLT2, or for use as aresearch, diagnostic, or therapeutic agent in accordance with thepresent invention.

The target segments of the present invention may be also be combinedwith their respective complementary antisense compounds of the presentinvention to form stabilized double-stranded (duplexed)oligonucleotides.

Such double stranded oligonucleotide moieties have been shown in the artto modulate target expression and regulate translation as well as RNAprocesssing via an antisense mechanism. Moreover, the double-strandedmoieties may be subject to chemical modifications (Fire et al., Nature,1998, 391, 806-811; Timmons and Fire, Nature 1998, 395, 854; Timmons etal., Gene, 2001, 263, 103-112; Tabara et al., Science, 1998, 282,430-431; Montgomery et al., Proc. Natl. Acad. Sci. USA, 1998, 95,15502-15507; Tuschl et al., Genes Dev., 1999, 13, 3191-3197; Elbashir etal., Nature, 2001, 411, 494-498; Elbashir et al., Genes Dev. 2001, 15,188-200). For example, such double-stranded moieties have been shown toinhibit the target by the classical hybridization of antisense strand ofthe duplex to the target, thereby triggering enzymatic degradation ofthe target (Tijsterman et al., Science, 2002, 295, 694-697).

The antisense compounds of the present invention can also be applied inthe areas of drug discovery and target validation. The present inventioncomprehends the use of the compounds and target segments identifiedherein in drug discovery efforts to elucidate relationships that existbetween SGLT2 and a disease state, phenotype, or condition. Thesemethods include detecting or modulating SGLT2 comprising contacting asample, tissue, cell, or organism with the compounds of the presentinvention, measuring the nucleic acid or protein level of SGLT2 and/or arelated phenotypic or chemical endpoint at some time after treatment,and optionally comparing the measured value to a non-treated sample orsample treated with a further compound of the invention. These methodscan also be performed in parallel or in combination with otherexperiments to determine the function of unknown genes for the processof target validation or to determine the validity of a particular geneproduct as a target for treatment or prevention of a particular disease,condition, or phenotype.

The antisense compounds of the present invention can be utilized fordiagnostics, therapeutics, prophylaxis and as research reagents andkits. Furthermore, antisense oligonucleotides, which are able to inhibitgene expression with exquisite specificity, are often used by those ofordinary skill to elucidate the function of particular genes or todistinguish between functions of various members of a biologicalpathway.

For use in kits and diagnostics, the compounds of the present invention,either alone or in combination with other compounds or therapeutics, canbe used as tools in differential and/or combinatorial analyses toelucidate expression patterns of a portion or the entire complement ofgenes expressed within cells and tissues.

As one nonlimiting example, expression patterns within cells or tissuestreated with one or more antisense compounds are compared to controlcells or tissues not treated with antisense compounds and the patternsproduced are analyzed for differential levels of gene expression as theypertain, for example, to disease association, signaling pathway,cellular localization, expression level, size, structure or function ofthe genes examined. These analyses can be performed on stimulated orunstimulated cells and in the presence or absence of other compoundswhich affect expression patterns.

Examples of methods of gene expression analysis known in the art includeDNA arrays or microarrays (Brazma and Vilo, FEBS Lett., 2000, 480,17-24; Celis, et al., FEBS Lett., 2000, 480, 2-16), SAGE (serialanalysis of gene expression)(Madden, et al., Drug Discov. Today, 2000,5, 415-425), READS (restriction enzyme amplification of digested cDNAs)(Prashar and Weissman, Methods Enzymol., 1999, 303, 258-72), TOGA (totalgene expression analysis) (Sutcliffe, et al., Proc. Natl. Acad. Sci.U.S.A., 2000, 97, 1976-81), protein arrays and proteomics (Celis, etal., FEBS Lett., 2000, 480, 2-16; Jungblut, et al., Electrophoresis,1999, 20, 2100-10), expressed sequence tag (EST) sequencing (Celis, etal., FEBS Lett., 2000, 480, 2-16; Larsson, et al., J. Biotechnol., 2000,80, 143-57), subtractive RNA fingerprinting (SuRF) (Fuchs, et al., Anal.Biochem., 2000, 286, 91-98; Larson, et al., Cytometry, 2000, 41,203-208), subtractive cloning, differential display (DD) (Jurecic andBelmont, Curr. Opin. Microbiol., 2000, 3, 316-21), comparative genomichybridization (Carulli, et al., J. Cell Biochem. Suppl., 1998, 31,286-96), FISH (fluorescent in situ hybridization) techniques (Going andGusterson, Eur. J. Cancer, 1999, 35, 1895-904) and mass spectrometrymethods (To, Comb. Chem. High Throughput Screen, 2000, 3, 235-41).

The antisense compounds of the invention are useful for research anddiagnostics, because these compounds hybridize to nucleic acids encodingSGLT2. For example, oligonucleotides that are shown to hybridize withsuch efficiency and under such conditions as disclosed herein as to beeffective SGLT2 inhibitors will also be effective primers or probesunder conditions favoring gene amplification or detection, respectively.These primers and probes are useful in methods requiring the specificdetection of nucleic acid molecules encoding SGLT2 and in theamplification of said nucleic acid molecules for detection or for use infurther studies of SGLT2. Hybridization of the antisenseoligonucleotides, particularly the primers and probes, of the inventionwith a nucleic acid encoding SGLT2 can be detected by means known in theart. Such means may include conjugation of an enzyme to theoligonucleotide, radiolabelling of the oligonucleotide or any othersuitable detection means. Kits using such detection means for detectingthe level of SGLT2 in a sample may also be prepared.

The specificity and sensitivity of antisense is also harnessed by thoseof skill in the art for therapeutic uses. Antisense compounds have beenemployed as therapeutic moieties in the treatment of disease states inanimals, including humans. Antisense oligonucleotide drugs, includingribozymes, have been safely and effectively administered to humans andnumerous clinical trials are presently underway. It is thus establishedthat antisense compounds can be useful therapeutic modalities that canbe configured to be useful in treatment regimes for the treatment ofcells, tissues and animals, especially humans.

For therapeutics, an animal, such as a human, suspected of having adisease or disorder which can be treated by modulating the expression ofSGLT2 is treated by administering antisense compounds in accordance withthis invention. For example, in one non-limiting embodiment, the methodscomprise the step of administering to the animal a therapeuticallyeffective amount of a SGLT2 inhibitor. The animal may or may not havealready been identifies as being in need of treatment. That is, theanimal may or may not have been diagnosed with a particular disease ordisorder. The SGLT2 inhibitors of the present invention effectivelyinhibit the activity of the SGLT2 protein or inhibit the expression ofthe SGLT2 protein. In some embodiments, the activity or expression ofSGLT2 in an animal or cell is inhibited by at least about 10%, by atleast about 20%, by at least about 30%, by at least about 40%, by atleast about 50%, by at least about 60%, by at least about 70%, by atleast about 75%, by at least about 80%, by at least about 85%, by atleast about 90%, by at least about 95%, by at least about 97%, by atleast about 99%, or by 100%.

For example, the reduction of the expression of SGLT2 may be measured inserum, adipose tissue, liver or any other body fluid, tissue or organ ofthe animal. The cells contained within the fluids, tissues or organsbeing analyzed can contain a nucleic acid molecule encoding SGLT2protein and/or the SGLT2 protein itself.

The antisense compounds of the invention can be utilized inpharmaceutical compositions by adding an effective amount of a compoundto a suitable pharmaceutically acceptable diluent or carrier. Use of thecompounds and methods of the invention may also be usefulprophylactically.

As is known in the art, a nucleoside is a base-sugar combination. Thebase portion of the nucleoside is normally a heterocyclic base sometimesreferred to as a “nucleobase” or simply a “base.” The two most commonclasses of such heterocyclic bases are the purines and the pyrimidines.Nucleotides are nucleosides that further include a phosphate groupcovalently linked to the sugar portion of the nucleoside. For thosenucleosides that include a pentofuranosyl sugar, the phosphate group canbe linked to either the 2′, 3′ or 5′ hydroxyl moiety of the sugar. Informing oligonucleotides, the phosphate groups covalently link adjacentnucleosides to one another to form a linear polymeric compound. In turn,the respective ends of this linear polymeric compound can be furtherjoined to form a circular compound, however, linear compounds aregenerally desired. In addition, linear compounds may have internalnucleobase complementarity and may therefore fold in a manner as toproduce a fully or partially double-stranded compound. Withinoligonucleotides, the phosphate groups are commonly referred to asforming the internucleoside backbone of the oligonucleotide. The normallinkage or backbone of RNA and DNA is a 3′ to 5′ phosphodiester linkage.

Modified Internucleoside Linkages (Backbones)

Specific examples of antisense compounds useful in this inventioninclude oligonucleotides containing modified backbones or non-naturalinternucleoside linkages. As defined in this specification,oligonucleotides having modified backbones include those that retain aphosphorus atom in the backbone and those that do not have a phosphorusatom in the backbone. For the purposes of this specification, and assometimes referenced in the art, modified oligonucleotides that do nothave a phosphorus atom in their internucleoside backbone can also beconsidered to be oligonucleosides.

Modified oligonucleotide backbones containing a phosphorus atom thereininclude, for example, phosphorothioates, chiral phosphorothioates,phosphorodithioates, phosphotriesters,aminoalkylphosphotriaminoalkylphosphotriesters, methyl and other alkylphosphonates including 3′-alkylene phosphonates, 5′-alkylenephosphonates and chiral phosphonates, phosphinates, phosphoramidatesincluding 3′-amino phosphoramidate and aminoalkylphosphoramidates,thionophosphoramidates, thionoalkylphosphonates,thionoalkylphosphotriesters, selenophosphates and boranophosphateshaving normal 3′-5′ linkages, 2′-5′ linked analogs of these, and thosehaving inverted polarity wherein one or more internucleotide linkages isa 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage. Oligonucleotides havinginverted polarity can comprise a single 3′ to 3′ linkage at the 3′-mostinternucleotide linkage i.e. a single inverted nucleoside residue whichmay be abasic (the nucleobase is missing or has a hydroxyl group inplace thereof). Various salts, mixed salts and free acid forms are alsoincluded.

Representative U.S. patents that teach the preparation of the abovephosphorus-containing linkages include, but are not limited to, U.S.Pat. No. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799;5,587,361; 5,194,599; 5,565,555; 5,527,899; 5,721,218; 5,672,697 and5,625,050.

Modified oligonucleotide backbones that do not include a phosphorus atomtherein can have backbones that are formed by short chain alkyl orcycloalkyl internucleoside linkages, mixed heteroatom and alkyl orcycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; riboacetyl backbones; alkene containingbackbones; sulfamate backbones; methyleneimino and methylenehydrazinobackbones; sulfonate and sulfonamide backbones; amide backbones; andothers having mixed N, O, S and CH₂ component parts.

Representative U.S. patents that teach the preparation of the aboveoligonucleosides include, but are not limited to, U.S. Pat. No.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439.

Modified Sugar and Internucleoside Linkages-Mimetics

In other antisense compounds, e.g., oligonucleotide mimetics, both thesugar and the internucleoside linkage (i.e. the backbone), of thenucleotide units are replaced with novel groups. The nucleobase unitsare maintained for hybridization with an appropriate target nucleicacid. One such compound, an oligonucleotide mimetic that has been shownto have excellent hybridization properties, is referred to as a peptidenucleic acid (PNA). In PNA compounds, the sugar-backbone of anoligonucleotide is replaced with an amide containing backbone, inparticular an aminoethylglycine backbone. The nucleobases are retainedand are bound directly or indirectly to aza nitrogen atoms of the amideportion of the backbone. Representative U.S. patents that teach thepreparation of PNA compounds include, but are not limited to, U.S. Pat.No. 5,539,082; 5,714,331; and 5,719,262. Further teaching of PNAcompounds can be found in Nielsen et al., Science, 1991, 254, 1497-1500.

Some embodiments of the invention include oligonucleotides withphosphorothioate backbones and oligonucleosides with heteroatombackbones, and in particular —CH₂—NH—O—CH₂—, —CH₂—N(CH₃)—O—CH₂— (knownas a methylene (methylimino) or MMI backbone), —CH₂—O—N(CH₃)—CH₂—,—CH₂—N(CH₃)—N(CH₃)—CH₂— and —O—N(CH₃)—CH₂—CH₂— (wherein the nativephosphodiester backbone is represented as —O—P—O—CH₂—) of the abovereferenced U.S. Pat. No. 5,489,677, and the amide backbones of the abovereferenced U.S. Pat. No. 5,602,240. Also suitable are oligonucleotideshaving morpholino backbone structures of the above-referenced U.S. Pat.No. 5,034,506.

Modified Sugars

Modified antisense compounds may also contain one or more substitutedsugar moieties. Antisense compounds, such as antisense oligonucleotides,can comprise one of the following at the 2′ position: OH; F; O—, S—, orN-alkyl; O—, S—, or N-alkenyl; O—, S— or N-alkynyl; or O-alkyl-O-alkyl,wherein the alkyl, alkenyl and alkynyl may be substituted orunsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyl and alkynyl. Alsosuitable are O((CH₂)_(n)O)_(m)CH₃, O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂,O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON((CH₂)_(n)CH₃)₂, where nand m are from 1 to about 10. Other oligonucleotides can comprise one ofthe following at the 2′ position: C₁ to C₁₀ lower alkyl, substitutedlower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl,SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂,heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino,substituted silyl, an RNA cleaving group, a reporter group, anintercalator, a group for improving the pharmacokinetic properties of anoligonucleotide, or a group for improving the pharmacodynamic propertiesof an oligonucleotide, and other substituents having similar properties.One modification includes 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃, also knownas 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta,1995, 78, 486-504) i.e., an alkoxyalkoxy group. Another modificationincludes 2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂ group, alsoknown as 2′-DMAOE, as described in examples hereinbelow, and2′-dimethylaminoethoxyethoxy (also known in the art as2′-O-dimethyl-amino-ethoxy-ethyl or 2′-DMAEOE), i.e.,2′-O—CH₂—O—CH₂—N(CH₃)₂, also described in examples hereinbelow.

Other modifications include 2′-methoxy (2′-O—CH₃), 2′-aminopropoxy(2′-OCH₂CH₂CH₂NH₂), 2′-allyl (2′-CH₂—CH═CH₂), 2′-O-allyl(2′-O—CH₂—CH═CH₂) and 2′-fluoro (2′-F). The 2′-modification may be inthe arabino (up) position or ribo (down) position. A preferred2′-arabino modification is 2′-F. Similar modifications may also be madeat other positions on the oligonucleotide, particularly the 3′ positionof the sugar on the 3′ terminal nucleotide or in 2′-5′ linkedoligonucleotides and the 5′ position of 5′ terminal nucleotide.Antisense compounds may also have sugar mimetics such as cyclobutylmoieties in place of the pentofuranosyl sugar. Representative U.S.patents that teach the preparation of such modified sugar structuresinclude, but are not limited to, U.S. Pat. No. 4,981,957; 5,118,800;5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785;5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300;5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; 5,792,747; and5,700,920.

Another modification of the sugar includes Locked Nucleic Acids (LNAs)in which the 2′-hydroxyl group is linked to the 3′ or 4′ carbon atom ofthe sugar ring, thereby forming a bicyclic sugar moiety. The linkage canbe a methylene (—CH₂—)_(n) group bridging the 2′ oxygen atom and the 4′carbon atom wherein n is 1 or 2. LNAs and preparation thereof aredescribed in WO 98/39352 and WO 99/14226.

Natural and Modified Nucleobases

Antisense compounds may also include nucleobase (often referred to inthe art as heterocyclic base or simply as “base”) modifications orsubstitutions. As used herein, “unmodified” or “natural” nucleobasesinclude the purine bases adenine (A) and guanine (G), and the pyrimidinebases thymine (T), cytosine (C) and uracil (U). Modified nucleobasesinclude other synthetic and natural nucleobases such as 5-methylcytosine(5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine,2-aminoadenine, 6-methyl and other alkyl derivatives of adenine andguanine, 2-propyl and other alkyl derivatives of adenine and guanine,2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil andcytosine, 5-propynyl (—C≡C—CH₃) uracil and cytosine and other alkynylderivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine,5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol,8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines,5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituteduracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine,2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further modifiednucleobases include tricyclic pyrimidines such as phenoxazinecytidine(1H-pyrimido(5,4-b)(1,4)benzoxazin-2(3H)-one), phenothiazinecytidine (1H-pyrimido(5,4-b)(1,4)benzothiazin-2(3H)-one), G-clamps suchas a substituted phenoxazine cytidine (e.g.9-(2-aminoethoxy)-H-pyrimido(5,4-b)(1,4)benzoxazin-2(3H)-one), carbazolecytidine (2H-pyrimido(4,5-b)indol-2-one), pyridoindole cytidine(H-pyrido(3′,2′:4,5)pyrrolo(2,3-d)pyrimidin-2-one). Modified nucleobasesmay also include those in which the purine or pyrimidine base isreplaced with other heterocycles, for example 7-deaza-adenine,7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobasesinclude those disclosed in U.S. Pat. No. 3,687,808, those disclosed inThe Concise Encyclopedia Of Polymer Science And Engineering, pages858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosedby Englisch et al., Angewandte Chemie, International Edition, 1991, 30,613, and those disclosed by Sanghvi, Y. S., Chapter 15, AntisenseResearch and Applications, pages 289-302, Crooke, S. T. and Lebleu, B.,ed., CRC Press, 1993. Certain of these nucleobases are particularlyuseful for increasing the binding affinity of the compounds of theinvention. These include 5-substituted pyrimidines, 6-azapyrimidines andN-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine,5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutionshave been shown to increase nucleic acid duplex stability by 0.6-1.2° C.and are presently suitable base substitutions, even more particularlywhen combined with 2′-O-methoxyethyl sugar modifications.

Representative U.S. patents that teach the preparation of certain of theabove noted modified nucleobases as well as other modified nucleobasesinclude, but are not limited to, the above noted U.S. Pat. No.3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302; 5,134,066;5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908;5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091;5,614,617; 5,645,985; 5,830,653; 5,763,588; 6,005,096; and 5,681,941,and 5,750,692.

Conjugates

Another modification of the antisense compounds of the inventioninvolves chemically linking to the antisense compound one or moremoieties or conjugates which enhance the activity, cellular distributionor cellular uptake of the oligonucleotide. These moieties or conjugatescan include conjugate groups covalently bound to functional groups suchas primary or secondary hydroxyl groups. Conjugate groups of theinvention include, but are not limited to, intercalators, reportermolecules, polyamines, polyamides, polyethylene glycols, polyethers,groups that enhance the pharmacodynamic properties of oligomers, andgroups that enhance the pharmacokinetic properties of oligomers. Typicalconjugate groups include, but are not limited to, cholesterols, lipids,phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone,acridine, fluoresceins, rhodamines, coumarins, and dyes. Groups thatenhance the pharmacodynamic properties, in the context of thisinvention, include groups that improve uptake, enhance resistance todegradation, and/or strengthen sequence-specific hybridization with thetarget nucleic acid. Groups that enhance the pharmacokinetic properties,in the context of this invention, include groups that improve uptake,distribution, metabolism or excretion of the compounds of the presentinvention. Representative conjugate groups are disclosed inInternational Patent Application PCT/US92/09196, filed Oct. 23, 1992,and U.S. Pat. No. 6,287,860. Conjugate moieties include, but are notlimited to, lipid moieties such as a cholesterol moiety, cholic acid, athioether, e.g., hexyl-S-tritylthiol, a thiocholesterol, an aliphaticchain, e.g., dodecandiol or undecyl residues, a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or apolyethylene glycol chain, or adamantane acetic acid, a palmityl moiety,or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety.Antisense compounds of the invention may also be conjugated to activedrug substances, for example, aspirin, warfarin, phenylbutazone,ibuprofen, suprofen, fenbufen, ketoprofen, (S)-(+)-pranoprofen,carprofen, dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid,folinic acid, a benzothiadiazide, chlorothiazide, a diazepine,indomethicin, a barbiturate, a cephalosporin, a sulfa drug, anantidiabetic, an antibacterial or an antibiotic. Oligonucleotide-drugconjugates and their preparation are described in U.S. patentapplication Ser. No. 09/334,130 (filed Jun. 15, 1999).

Representative U.S. patents that teach the preparation of sucholigonucleotide conjugates include, but are not limited to, U.S. Pat.Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730;5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124;5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718;5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737;4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830;5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022;5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098;5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667;5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371;5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941.

Chimeric Compounds

It is not necessary for all positions in a given compound to beuniformly modified, and in fact more than one of the aforementionedmodifications may be incorporated in a single compound or even at asingle nucleoside within an oligonucleotide.

The present invention also includes antisense compounds which arechimeric compounds. “Chimeric” antisense compounds or “chimeras,” in thecontext of this invention, are antisense compounds, particularlyoligonucleotides, which contain two or more chemically distinct regions,each made up of at least one monomer unit, i.e., a nucleotide in thecase of an oligonucleotide compound. Chimeric antisense oligonucleotidesare thus a form of antisense compound. These oligonucleotides typicallycontain at least one region wherein the oligonucleotide is modified soas to confer upon the oligonucleotide increased resistance to nucleasedegradation, increased cellular uptake, increased stability and/orincreased binding affinity for the target nucleic acid. An additionalregion of the oligonucleotide may serve as a substrate for enzymescapable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNAseH is a cellular endonuclease which cleaves the RNA strand of an RNA:DNAduplex. Activation of RNase H, therefore, results in cleavage of the RNAtarget, thereby greatly enhancing the efficiency ofoligonucleotide-mediated inhibition of gene expression. The cleavage ofRNA:RNA hybrids can, in like fashion, be accomplished through theactions of endoribonucleases, such as RNAseL which cleaves both cellularand viral RNA. Cleavage of the RNA target can be routinely detected bygel electrophoresis and, if necessary, associated nucleic acidhybridization techniques known in the art.

Chimeric antisense compounds of the invention may be formed as compositestructures of two or more oligonucleotides, modified oligonucleotides,oligonucleosides and/or oligonucleotide mimetics as described above.Such compounds have also been referred to in the art as hybrids orgapmers. Representative U.S. patents that teach the preparation of suchhybrid structures include, but are not limited to, U.S. Pat. Nos.5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711;5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922.

The compounds of the invention may also be admixed, encapsulated,conjugated or otherwise associated with other molecules, moleculestructures or mixtures of compounds, as for example, liposomes,receptor-targeted molecules, oral, rectal, topical or otherformulations, for assisting in uptake, distribution and/or absorption.Representative U.S. patents that teach the preparation of such uptake,distribution and/or absorption-assisting formulations include, but arenot limited to, U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016;5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721;4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170;5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854;5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948;5,580,575; and 5,595,756.

The antisense compounds of the invention encompass any pharmaceuticallyacceptable salts, esters, or salts of such esters, or any other compoundwhich, upon administration to an animal, including a human, is capableof providing (directly or indirectly) the biologically active metaboliteor residue thereof.

The term “pharmaceutically acceptable salts” refers to physiologicallyand pharmaceutically acceptable salts of the compounds of the invention:i.e., salts that retain the desired biological activity of the parentcompound and do not impart undesired toxicological effects thereto. Foroligonucleotides, examples of pharmaceutically acceptable salts andtheir uses are further described in U.S. Pat. No. 6,287,860. Potassiumand sodium salts are typical salts.

The present invention also includes pharmaceutical compositions andformulations which include the antisense compounds of the invention. Thepharmaceutical compositions of the present invention may be administeredin a number of ways depending upon whether local or systemic treatmentis desired and upon the area to be treated. Administration may betopical (including ophthalmic and to mucous membranes including vaginaland rectal delivery), pulmonary, e.g., by inhalation or insufflation ofpowders or aerosols, including by nebulizer; intratracheal, intranasal,epidermal and transdermal), oral or parenteral. Parenteraladministration includes intravenous, intraarterial, subcutaneous,intraperitoneal or intramuscular injection or infusion; or intracranial,e.g., intrathecal or intraventricular, administration. Oligonucleotideswith at least one 2′-O-methoxyethyl modification are believed to beparticularly useful for oral administration. Pharmaceutical compositionsand formulations for topical administration may include transdermalpatches, ointments, lotions, creams, gels, drops, suppositories, sprays,liquids and powders. Conventional pharmaceutical carriers, aqueous,powder or oily bases, thickeners and the like may be necessary ordesirable. Coated condoms, gloves and the like may also be useful.

The pharmaceutical formulations of the present invention, which mayconveniently be presented in unit dosage form, may be prepared accordingto conventional techniques well known in the pharmaceutical industry.Such techniques include the step of bringing into association the activeingredients with the pharmaceutical carrier(s) or excipient(s). Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

The compositions of the present invention may be formulated into any ofmany possible dosage forms such as, but not limited to, tablets,capsules, gel capsules, liquid syrups, soft gels, suppositories, andenemas. The compositions of the present invention may also be formulatedas suspensions in aqueous, non-aqueous or mixed media. Aqueoussuspensions may further contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

Pharmaceutical compositions of the present invention include, but arenot limited to, solutions, emulsions, foams and liposome-containingformulations. The pharmaceutical compositions and formulations of thepresent invention may comprise one or more penetration enhancers,carriers, excipients or other active or inactive ingredients.

Emulsions are typically heterogenous systems of one liquid dispersed inanother in the form of droplets usually exceeding 0.1 μm in diameter.Emulsions may contain additional components in addition to the dispersedphases, and the active drug which may be present as a solution in eitherthe aqueous phase, oily phase or itself as a separate phase.Microemulsions are included as an embodiment of the present invention.Emulsions and their uses are well known in the art and are furtherdescribed in U.S. Pat. No. 6,287,860.

Formulations of the present invention include liposomal formulations. Asused in the present invention, the term “liposome” means a vesiclecomposed of amphiphilic lipids arranged in a spherical bilayer orbilayers. Liposomes are unilamellar or multilamellar vesicles which havea membrane formed from a lipophilic material and an aqueous interiorthat contains the composition to be delivered. Cationic liposomes arepositively charged liposomes which are believed to interact withnegatively charged DNA molecules to form a stable complex. Liposomesthat are pH-sensitive or negatively-charged are believed to entrap DNArather than complex with it. Both cationic and noncationic liposomeshave been used to deliver DNA to cells.

Liposomes also include “sterically stabilized” liposomes, a term which,as used herein, refers to liposomes comprising one or more specializedlipids that, when incorporated into liposomes, result in enhancedcirculation lifetimes relative to liposomes lacking such specializedlipids. Examples of sterically stabilized liposomes are those in whichpart of the vesicle-forming lipid portion of the liposome comprises oneor more glycolipids or is derivatized with one or more hydrophilicpolymers, such as a polyethylene glycol (PEG) moiety. Liposomes andtheir uses are further described in U.S. Pat. No. 6,287,860.

The pharmaceutical formulations and compositions of the presentinvention may also include surfactants. The use of surfactants in drugproducts, formulations and in emulsions is well known in the art.Surfactants and their uses are further described in U.S. Pat. No.6,287,860.

In one embodiment, the present invention employs various penetrationenhancers to effect the efficient delivery of nucleic acids,particularly oligonucleotides. In addition to aiding the diffusion ofnon-lipophilic drugs across cell membranes, penetration enhancers alsoenhance the permeability of lipophilic drugs. Penetration enhancers maybe classified as belonging to one of five broad categories, i.e.,surfactants, fatty acids, bile salts, chelating agents, andnon-chelating non-surfactants. Penetration enhancers and their uses arefurther described in U.S. Pat. No. 6,287,860.

One of skill in the art will recognize that formulations are routinelydesigned according to their intended use, i.e. route of administration.

Formulations for topical administration include those in which theoligonucleotides of the invention are in admixture with a topicaldelivery agent such as lipids, liposomes, fatty acids, fatty acidesters, steroids, chelating agents and surfactants. Lipids and liposomesinclude neutral (e.g. dioleoylphosphatidyl DOPE ethanolamine,dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline)negative (e.g. dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g.dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidylethanolamine DOTMA).

For topical or other administration, oligonucleotides of the inventionmay be encapsulated within liposomes or may form complexes thereto, inparticular to cationic liposomes. Alternatively, oligonucleotides may becomplexed to lipids, in particular to cationic lipids. Fatty acids andesters, pharmaceutically acceptable salts thereof, and their uses arefurther described in U.S. Pat. No. 6,287,860. Topical formulations aredescribed in detail in U.S. patent application Ser. No. 09/315,298 filedon May 20, 1999.

Compositions and formulations for oral administration include powders orgranules, microparticulates, nanoparticulates, suspensions or solutionsin water or non-aqueous media, capsules, gel capsules, sachets, tabletsor minitablets. Thickeners, flavoring agents, diluents, emulsifiers,dispersing aids or binders may be desirable. Suitable oral formulationsare those in which oligonucleotides of the invention are administered inconjunction with one or more penetration enhancers surfactants andchelators. Surfactants include fatty acids and/or esters or saltsthereof, bile acids and/or salts thereof. Bile acids/salts and fattyacids and their uses are further described in U.S. Pat. No. 6,287,860.Also suitable are combinations of penetration enhancers, for example,fatty acids/salts in combination with bile acids/salts. A particularlysuitable combination is the sodium salt of lauric acid, capric acid andUDCA. Further penetration enhancers include polyoxyethylene-9-laurylether, polyoxyethylene-20-cetyl ether. Oligonucleotides of the inventionmay be delivered orally, in granular form including sprayed driedparticles, or complexed to form micro or nanoparticles. Oligonucleotidecomplexing agents and their uses are further described in U.S. Pat. No.6,287,860. Oral formulations for oligonucleotides and their preparationare described in detail in U.S. applications Ser. No. 09/108,673 (filedJul. 1, 1998), Ser. No. 09/315,298 (filed May 20, 1999) and Ser. No.10/071,822, filed Feb. 8, 2002 and published as U.S. Application No.2003-0027780.

Compositions and formulations for parenteral, intrathecal orintraventricular administration may include sterile aqueous solutionswhich may also contain buffers, diluents and other suitable additivessuch as, but not limited to, penetration enhancers, carrier compoundsand other pharmaceutically acceptable carriers or excipients.

Certain embodiments of the invention provide pharmaceutical compositionscontaining one or more oligomeric compounds and one or more otherchemotherapeutic agents which function by a non-antisense mechanism.Examples of such chemotherapeutic agents include, but are not limitedto, cancer chemotherapeutic drugs such as daunorubicin, daunomycin,dactinomycin, doxorubicin, epirubicin, idarubicin, esorubicin,bleomycin, mafosfamide, ifosfamide, cytosine arabinoside,bis-chloroethylnitrosurea, busulfan, mitomycin C, actinomycin D,mithramycin, prednisone, hydroxyprogesterone, testosterone, tamoxifen,dacarbazine, procarbazine, hexamethylmelamine, pentamethylmelamine,mitoxantrone, amsacrine, chlorambucil, methylcyclohexylnitrosurea,nitrogen mustards, melphalan, cyclophosphamide, 6-mercaptopurine,6-thioguanine, cytarabine, 5-azacytidine, hydroxyurea, deoxycoformycin,4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU),5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol,vincristine, vinblastine, etoposide (VP-16), trimetrexate, irinotecan,topotecan, gemcitabine, teniposide, cisplatin and diethylstilbestrol(DES). When used with the compounds of the invention, suchchemotherapeutic agents may be used individually (e.g., 5-FU andoligonucleotide), sequentially (e.g., 5-FU and oligonucleotide for aperiod of time followed by MTX and oligonucleotide), or in combinationwith one or more other such chemotherapeutic agents (e.g., 5-FU, MTX andoligonucleotide, or 5-FU, radiotherapy and oligonucleotide).Anti-inflammatory drugs including, but not limited to, nonsteroidalanti-inflammatory drugs and corticosteroids, and antiviral drugsincluding, but not limited to, ribivirin, vidarabine, acyclovir andganciclovir, may also be combined in compositions of the invention.Combinations of antisense compounds and other non-antisense drugs arealso within the scope of this invention. Two or more combined compoundsmay be used together or sequentially.

In another related embodiment, compositions of the invention may containone or more antisense compounds, particularly oligonucleotides, targetedto a first nucleic acid and one or more additional antisense compoundstargeted to a second nucleic acid target. Alternatively, compositions ofthe invention may contain two or more antisense compounds targeted todifferent regions of the same nucleic acid target. Numerous examples ofantisense compounds are known in the art. Two or more combined compoundsmay be used together or sequentially.

The formulation of therapeutic compositions and their subsequentadministration (dosing) is believed to be within the skill of those inthe art. Dosing is dependent on severity and responsiveness of thedisease state to be treated, with the course of treatment lasting fromseveral days to several months, or until a cure is effected or adiminution of the disease state is achieved. Optimal dosing schedulescan be calculated from measurements of drug accumulation in the body ofthe patient. Persons of ordinary skill can easily determine optimumdosages, dosing methodologies and repetition rates. Optimum dosages mayvary depending on the relative potency of individual oligonucleotides,and can generally be estimated based on EC₅₀s found to be effective inin vitro and in vivo animal models. In general, dosage is from 0.01 μgto 100 g per kg of body weight, from 0.1 μg to 10 g per kg of bodyweight, from 1 μg to 1 g per kg of body weight, from 10 μg to 100 mg perkg of body weight, from 100 μg to 10 mg per kg of body weight, or from100 μg to 1 mg per kg of body weight, and may be given once or moredaily, weekly, monthly or yearly, or even once every 2 to 20 years.Persons of ordinary skill in the art can easily estimate repetitionrates for dosing based on measured residence times and concentrations ofthe drug in bodily fluids or tissues. Following successful treatment, itmay be desirable to have the patient undergo maintenance therapy toprevent the recurrence of the disease state, wherein the oligonucleotideis administered in maintenance doses, ranging from 0.0001 μg to 100 gper kg of body weight, once or more daily, to once every 20 years.

In order that the invention disclosed herein may be more efficientlyunderstood, examples are provided below. It should be understood thatthese examples are for illustrative purposes only and are not to beconstrued as limiting the invention in any manner. Throughout theseexamples, molecular cloning reactions, and other standard recombinantDNA techniques, were carried out according to methods described inManiatis et al., Molecular Cloning—A Laboratory Manual, 2nd ed., ColdSpring Harbor Press (1989), using commercially available reagents,except where otherwise noted.

EXAMPLES Example 1 Synthesis of Nucleoside Phosphoramidites

The following compounds, including amidites and their intermediates wereprepared as described in U.S. Pat. No. 6,426,220 and published PCT WO02/36743; 5′-O-Dimethoxytrityl-thymidine intermediate for 5-methyl dCamidite, 5′-O-Dimethoxytrityl-2′-deoxy-5-methylcytidine intermediate for5-methyl-dC amidite,5′-O-Dimethoxytrityl-2′-deoxy-N4-benzoyl-5-methylcytidine penultimateintermediate for 5-methyl dC amidite,(5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-deoxy-N⁴-benzoyl-5-methylcytidin-3′-O-yl)-2-cyanoethyl-N,N-diisopropylphosphoramidite(5-methyl dC amidite), 2′-Fluorodeoxyadenosine, 2′-Fluorodeoxyguanosine,2′-Fluorouridine, 2′-Fluorodeoxycytidine, 2′-O-(2-Methoxyethyl) modifiedamidites, 2′-O-(2-methoxyethyl)-5-methyluridine intermediate,5′-O-DMT-2′-O-(2-methoxyethyl)-5-methyluridine penultimate intermediate,(5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-5-methyluridin-3′-O-yl)-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE T amidite),5′-O-Dimethoxytrityl-2′-O-(2-methoxyethyl)-5-methylcytidineintermediate,5′-O-dimethoxytrityl-2′-0-(2-methoxyethyl)-N⁴-benzoyl-5-methyl-cytidinepenultimate intermediate,(5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁴-benzoyl-5-methylcytidin-3′-O-yl)-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE 5-Me—C amidite),(5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁶-benzoyladenosin-3′-O-yl)-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE A amdite),(5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁴-isobutyrylguanosin-3′-O-yl)-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE G amidite), 2′-O-(Aminooxyethyl) nucleoside amidites and2′-O-(dimethylaminooxyethyl) nucleoside amidites,2′-(Dimethylaminooxyethoxy) nucleoside amidites,5′-O-tert-Butyldiphenylsilyl-O²-2′-anhydro-5-methyluridine,5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine,2′-O-((2-phthalimidoxy)ethyl)-5′-t-butyldiphenylsilyl-5-methyluridine,5′-O-tert-butyldiphenylsilyl-2′-O-((2-formadoximinooxy)ethyl)-5-methyluridine,5′-O-tert-Butyldiphenylsilyl-2′-O-(N,Ndimethylaminooxyethyl)-5-methyluridine,2′-O-(dimethylaminooxyethyl)-5-methyluridine,5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine,5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-((2-cyanoethyl)-N,N-diisopropylphosphoramidite),2′-(Aminooxyethoxy) nucleoside amidites,N2-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-((2-cyanoethyl)-N,N-diisopropylphosphoramidite),2′-dimethylaminoethoxyethoxy (2′-DMAEOE) nucleoside amidites,2′-O-(2(2-N,N-dimethylaminoethoxy)ethyl)-5-methyl uridine,5′-O-dimethoxytrityl-2′-O-(2(2-N,N-dimethylaminoethoxy)-ethyl))-5-methyluridine and5′-O-Dimethoxytrityl-2′-O-(2(2-N,N-dimethylaminoethoxy)-ethyl))-5-methyluridine-3′-O-(cyanoethyl-N,N-diisopropyl)phosphoramidite.

Example 2 Oligonucleotide and Oligonucleoside Synthesis

The antisense compounds used in accordance with this invention may beconveniently and routinely made through the well-known technique ofsolid phase synthesis. Equipment for such synthesis is sold by severalvendors including, for example, Applied Biosystems (Foster City,Calif.). Any other means for such synthesis known in the art mayadditionally or alternatively be employed. It is well known to usesimilar techniques to prepare oligonucleotides such as thephosphorothioates and alkylated derivatives.

Oligonucleotides:

Unsubstituted and substituted phosphodiester (P═O) oligonucleotides aresynthesized on an automated DNA synthesizer (Applied Biosystems model394) using standard phosphoramidite chemistry with oxidation by iodine.

Phosphorothioates (P═S) are synthesized similar to phosphodiesteroligonucleotides with the following exceptions: thiation was effected byutilizing a 10% w/v solution of 3,H-1,2-benzodithiole-3-one 1,1 -dioxidein acetonitrile for the oxidation of the phosphite linkages. Thethiation reaction step time was increased to 180 sec and preceded by thenormal capping step. After cleavage from the CPG column and deblockingin concentrated ammonium hydroxide at 55° C. (12-16 hr), theoligonucleotides were recovered by precipitating with >3 volumes ofethanol from a 1 M NH₄OAc solution. Phosphinate oligonucleotides areprepared as described in U.S. Pat. No. 5,508,270.

Alkyl phosphonate oligonucleotides are prepared as described in U.S.Pat. No. 4,469,863.

3′-Deoxy-3′-methylene phosphonate oligonucleotides are prepared asdescribed in U.S. Pat. Nos. 5,610,289 or 5,625,050.

Phosphoramidite oligonucleotides are prepared as described in U.S. Pat.No. 5,256,775 or U.S. Pat. No. 5,366,878.

Alkylphosphonothioate oligonucleotides are prepared as described inpublished PCT applications PCT/US94/00902 and PCT/US93/06976 (publishedas WO 94/17093 and WO 94/02499, respectively).

3′-Deoxy-3′-amino phosphoramidate oligonucleotides are prepared asdescribed in U.S. Pat. No. 5,476,925.

Phosphotriester oligonucleotides are prepared as described in U.S. Pat.No. 5,023,243.

Borano phosphate oligonucleotides are prepared as described in U.S. Pat.Nos. 5,130,302 and 5,177,198.

Oligonucleosides:

Methylenemethylimino linked oligonucleosides, also identified as MMIlinked oligonucleosides, methylenedimethylhydrazo linkedoligonucleosides, also identified as MDH linked oligonucleosides, andmethylenecarbonylamino linked oligonucleosides, also identified asamide-3 linked oligonucleosides, and methyleneaminocarbonyl linkedoligo-nucleosides, also identified as amide-4 linked oligonucleosides,as well as mixed backbone compounds having, for instance, alternatingMMI and P═O or P═S linkages are prepared as described in U.S. Pat. Nos.5,378,825, 5,386,023, 5,489,677, 5,602,240 and 5,610,289.

Formacetal and thioformacetal linked oligonucleosides are prepared asdescribed in U.S. Pat. Nos. 5,264,562 and 5,264,564.

Ethylene oxide linked oligonucleosides are prepared as described in U.S.Pat. No. 5,223,618.

Example 3 RNA Synthesis

In general, RNA synthesis chemistry is based on the selectiveincorporation of various protecting groups at strategic intermediaryreactions. Although one of ordinary skill in the art will understand theuse of protecting groups in organic synthesis, a useful class ofprotecting groups includes silyl ethers. In particular bulky silylethers are used to protect the 5′-hydroxyl in combination with anacid-labile orthoester protecting group on the 2′-hydroxyl. This set ofprotecting groups is then used with standard solid-phase synthesistechnology. It is important to lastly remove the acid labile orthoesterprotecting group after all other synthetic steps. Moreover, the earlyuse of the silyl protecting groups during synthesis ensures facileremoval when desired, without undesired deprotection of 2′ hydroxyl.

Following this procedure for the sequential protection of the5′-hydroxyl in combination with protection of the 2′-hydroxyl byprotecting groups that are differentially removed and are differentiallychemically labile, RNA oligonucleotides were synthesized.

RNA oligonucleotides are synthesized in a stepwise fashion. Eachnucleotide is added sequentially (3′- to 5′-direction) to a solidsupport-bound oligonucleotide. The first nucleoside at the 3′-end of thechain is covalently attached to a solid support. The nucleotideprecursor, a ribonucleoside phosphoramidite, and activator are added,coupling the second base onto the 5′-end of the first nucleoside. Thesupport is washed and any unreacted 5′-hydroxyl groups are capped withacetic anhydride to yield 5′-acetyl moieties. The linkage is thenoxidized to the more stable and ultimately desired P(V) linkage. At theend of the nucleotide addition cycle, the 5′-silyl group is cleaved withfluoride. The cycle is repeated for each subsequent nucleotide.

Following synthesis, the methyl protecting groups on the phosphates arecleaved in 30 minutes utilizing 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate (S₂Na₂)in DMF. The deprotection solution is washed from the solid support-boundoligonucleotide using water. The support is then treated with 40%methylamine in water for 10 minutes at 55° C. This releases the RNAoligonucleotides into solution, deprotects the exocyclic amines, andmodifies the 2′-groups. The oligonucleotides can be analyzed by anionexchange HPLC at this stage.

The 2′-orthoester groups are the last protecting groups to be removed.The ethylene glycol monoacetate orthoester protecting group developed byDharmacon Research, Inc. (Lafayette, Colo.), is one example of a usefulorthoester protecting group which, has the following importantproperties. It is stable to the conditions of nucleoside phosphoramiditesynthesis and oligonucleotide synthesis. However, after oligonucleotidesynthesis the oligonucleotide is treated with methylamine which not onlycleaves the oligonucleotide from the solid support but also removes theacetyl groups from the orthoesters. The resulting 2-ethyl-hydroxylsubstituents on the orthoester are less electron withdrawing than theacetylated precursor. As a result, the modified orthoester becomes morelabile to acid-catalyzed hydrolysis. Specifically, the rate of cleavageis approximately 10 times faster after the acetyl groups are removed.Therefore, this orthoester possesses sufficient stability in order to becompatible with oligonucleotide synthesis and yet, when subsequentlymodified, permits deprotection to be carried out under relatively mildaqueous conditions compatible with the final RNA oligonucleotideproduct.

Additionally, methods of RNA synthesis are well known in the art(Scaringe, S. A. Ph.D. Thesis, University of Colorado, 1996; Scaringe,S. A., et al., J. Am. Chem. Soc., 1998, 120, 11820-11821; Matteucci, M.D. and Caruthers, M. H. J. Am. Chem. Soc., 1981, 103, 3185-3191;Beaucage, S. L. and Caruthers, M. H. Tetrahedron Lett., 1981, 22,1859-1862; Dahl, B. J., et al., Acta Chem. Scand,. 1990, 44, 639-641;Reddy, M. P., et al., Tetrahedrom Lett., 1994, 25, 4311-4314; Wincott,F. et al., Nucleic Acids Res., 1995, 23, 2677-2684; Griffin, B. E., etal., Tetrahedron, 1967, 23, 2301-2313; Griffin, B. E., et al.,Tetrahedron, 1967, 23, 2315-2331).

RNA antisense compounds (RNA oligonucleotides) of the present inventioncan be synthesized by the methods herein or purchased from DharmaconResearch, Inc (Lafayette, Colo.). Once synthesized, complementary RNAantisense compounds can then be annealed by methods known in the art toform double stranded (duplexed) antisense compounds. For example,duplexes can be formed by combining 30 μl of each of the complementarystrands of RNA oligonucleotides (50 uM RNA oligonucleotide solution) and15 μl of 5× annealing buffer (100 mM potassium acetate, 30 mM HEPES-KOHpH 7.4, 2 mM magnesium acetate) followed by heating for 1 minute at 90°C., then 1 hour at 37° C. The resulting duplexed antisense compounds canbe used in kits, assays, screens, or other methods to investigate therole of a target nucleic acid, or for diagnostic or therapeuticpurposes.

Example 4 Synthesis of Chimeric Compounds

Chimeric oligonucleotides, oligonucleosides or mixedoligonucleotides/oligonucleosides of the invention can be of severaldifferent types. These include a first type wherein the “gap” segment oflinked nucleosides is positioned between 5′ and 3′ “wing” segments oflinked nucleosides and a second “open end” type wherein the “gap”segment is located at either the 3′ or the 5′ terminus of the oligomericcompound. Oligonucleotides of the first type are also known in the artas “gapmers” or gapped oligonucleotides. Oligonucleotides of the secondtype are also known in the art as “hemimers” or “wingmers”.

(2′-O—Me)-(2′-deoxy)-(2′O—Me) Chimeric Phosphorothioate Oligonucleotides

Chimeric oligonucleotides having 2′-O-alkyl phosphorothioate and2′-deoxy phosphorothioate oligonucleotide segments are synthesized usingan Applied Biosystems automated DNA synthesizer Model 394, as above.Oligonucleotides are synthesized using the automated synthesizer and2′-deoxy-5′-dimethoxytrityl-3′-O-phosphoramidite for the DNA portion and5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite for 5′ and 3′ wings.The standard synthesis cycle is modified by incorporating coupling stepswith increased reaction times for the5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite. The fully protectedoligonucleotide is cleaved from the support and deprotected inconcentrated ammonia (NH₄OH) for 12-16 hr at 55° C. The deprotectedoligo is then recovered by an appropriate method (precipitation, columnchromatography, volume reduced in vacuo and analyzedspetrophotometrically for yield and for purity by capillaryelectrophoresis and by mass spectrometry.

(2 ′-O-(2-Methoxyethyl))-(2′-deoxy)-(2′-O-(Methoxyethyl)) ChimericPhosphorothioate Oligonucleotides

(2′-O-(2-methoxyethyl))-(2′-deoxy)-(2′-O-(methoxyethyl)) chimericphosphorothioate oligonucleotides were prepared as per the procedureabove for the 2′-O-methyl chimeric oligonucleotide, with thesubstitution of 2′-O-(methoxyethyl) amidites for the 2′-O-methylamidites.

(2 ′-O-(2-Methoxyethyl)Phosphodiester)-(2 ′-deoxyPhosphorothioate)-(2′-O-(2-Methoxyethyl) Phosphodiester) ChimericOligonucleotides

(2′-O-(2-methoxyethyl phosphodiester)-(2′-deoxyphosphorothioate)-(2′-O-(methoxyethyl) phosphodiester) chimericoligonucleotides are prepared as per the above procedure for the2′-O-methyl chimeric oligonucleotide with the substitution of2′-O-(methoxyethyl) amidites for the 2′-O-methyl amidites, oxidationwith iodine to generate the phosphodiester internucleotide linkageswithin the wing portions of the chimeric structures and sulfurizationutilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) togenerate the phosphorothioate internucleotide linkages for the centergap.

Other chimeric oligonucleotides, chimeric oligonucleosides and mixedchimeric oligonucleotides/oligonucleosides are synthesized according toU.S. Pat. No. 5,623,065, herein incorporated by reference.

Example 5 Design and Screening of Duplexed Antisense Compounds TargetingSGLT2

In accordance with the present invention, a series of nucleic acidduplexes comprising the antisense compounds of the present invention andtheir complements can be designed to target SGLT2. The nucleobasesequence of the antisense strand of the duplex comprises at least an8-nucleobase portion of an oligonucleotide in Table 1. The ends of thestrands may be modified by the addition of one or more natural ormodified nucleobases to form an overhang. The sense strand of the dsRNAis then designed and synthesized as the complement of the antisensestrand and may also contain modifications or additions to eitherterminus. For example, in one embodiment, both strands of the dsRNAduplex would be complementary over the central nucleobases, each havingoverhangs at one or both termini.

For example, a duplex comprising an antisense strand having the sequenceCGAGAGGCGGACGGGACCG (SEQ ID NO: 268) and having a two-nucleobaseoverhang of deoxythymidine(dT) would have the following structure:  cgagaggcggacgggaccgTT Antisense (SEQ ID NO:269)   |||||||||||||||||||Strand TTgctctccgcctgccctggc Comple- (SEQ ID NO:270) ment

In another embodiment, a duplex comprising an antisense strand havingthe same sequence CGAGAGGCGGACGGGACCG (SEQ ID NO: 268) may be preparedwith blunt ends (no single stranded overhang) as shown:cgagaggcggacgggaccg Antisense ||||||||||||||||||| Strandgctctccgcctgccctggc Complement (SEQ ID NO:271)

RNA strands of the duplex can be synthesized by methods disclosed hereinor purchased from Dharmacon Research Inc., (Lafayette, Colo.). Oncesynthesized, the complementary strands are annealed. The single strandsare aliquoted and diluted to a concentration of 50 μM. Once diluted, 30μL of each strand is combined with 15 μL of a 5× solution of annealingbuffer. The final concentration of said buffer is 100 mM potassiumacetate, 30 mM HEPES-KOH pH 7.4, and 2 mM magnesium acetate. The finalvolume is 75 μL. This solution is incubated for 1 minute at 90° C. andthen centrifuged for 15 seconds. The tube is allowed to sit for 1 hourat 37° C. at which time the dsRNA duplexes are used in experimentation.The final concentration of the dsRNA duplex is 20 μM. This solution canbe stored frozen (−20° C.) and freeze-thawed up to 5 times.

Once prepared, the duplexed antisense compounds are evaluated for theirability to modulate SGLT2 expression.

When cells reached 80% confluency, they are treated with duplexedantisense compounds of the invention. For cells grown in 96-well plates,wells are washed once with 200 μL OPTI-MEM-1 reduced-serum medium (GibcoBRL) and then treated with 130 μL of OPTI-MEM-1 containing 12 μg/mLLIPOFECTIN (Gibco BRL) and the desired duplex antisense compound at afinal concentration of 200 mM. After 5 hours of treatment, the medium isreplaced with fresh medium. Cells are harvested 16 hours aftertreatment, at which time RNA is isolated and target reduction measuredby RT-PCR.

Example 6 Oligonucleotide Isolation

After cleavage from the controlled pore glass solid support anddeblocking in concentrated ammonium hydroxide at 55° C. for 12-16 hours,the oligonucleotides or oligonucleosides are recovered by precipitationout of 1 M NH₄OAc with >3 volumes of ethanol. Synthesizedoligonucleotides were analyzed by electrospray mass spectroscopy(molecular weight determination) and by capillary gel electrophoresisand judged to be at least 70% full length material. The relative amountsof phosphorothioate and phosphodiester linkages obtained in thesynthesis was determined by the ratio of correct molecular weightrelative to the −16 amu product (±32 ±48). For some studiesoligonucleotides were purified by HPLC, as described by Chiang et al.,J. Biol. Chem. 1991, 266, 18162-18171. Results obtained withHPLC-purified material were similar to those obtained with non-HPLCpurified material.

Example 7 Oligonucleotide Synthesis—96 Well Plate Format

Oligonucleotides were synthesized via solid phase P(III) phosphoramiditechemistry on an automated synthesizer capable of assembling 96 sequencessimultaneously in a 96-well format. Phosphodiester internucleotidelinkages were afforded by oxidation with aqueous iodine.Phosphorothioate internucleotide linkages were generated bysulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide(Beaucage Reagent) in anhydrous acetonitrile. Standard base-protectedbeta-cyanoethyl-diiso-propyl phosphoramidites were purchased fromcommercial vendors (e.g. PE-Applied Biosystems, Foster City, Calif., orPharmacia, Piscataway, N.J.). Non-standard nucleosides are synthesizedas per standard or patented methods. They are utilized as base protectedbeta-cyanoethyldiisopropyl phosphoramidites.

Oligonucleotides were cleaved from support and deprotected withconcentrated NH₄OH at elevated temperature (55-60° C.) for 12-16 hoursand the released product then dried in vacuo. The dried product was thenre-suspended in sterile water to afford a master plate from which allanalytical and test plate samples are then diluted utilizing roboticpipettors.

Example 8 Oligonucleotide Analysis—96-Well Plate Format

The concentration of oligonucleotide in each well was assessed bydilution of samples and ]UV absorption spectroscopy. The full-lengthintegrity of the individual products was evaluated by capillaryelectrophoresis (CE) in either the 96-well format (Beckman P/ACE™ MDQ)or, for individually prepared samples, on a commercial CE apparatus(e.g., Beckman P/ACE™ 5000, ABI 270). Base and backbone composition wasconfirmed by mass analysis of the compounds utilizing electrospray-massspectroscopy. All assay test plates were diluted from the master plateusing single and multi-channel robotic pipettors. Plates were judged tobe acceptable if at least 85% of the compounds on the plate were atleast 85% full length.

Example 9 Cell Culture and Oligonucleotide Treatment

The effect of antisense compounds on target nucleic acid expression canbe tested in any of a variety of cell types provided that the targetnucleic acid is present at measurable levels. This can be routinelydetermined using, for example, PCR or Northern blot analysis. Thefollowing cell types are provided for illustrative purposes, but othercell types can be routinely used, provided that the target is expressedin the cell type chosen. This can be readily determined by methodsroutine in the art, for example Northern blot analysis, ribonucleaseprotection assays, or RT-PCR.

T-24 Cells:

The human transitional cell bladder carcinoma cell line T-24 wasobtained from the American Type Culture Collection (ATCC) (Nanassas,Va.). T-24 cells were routinely cultured in complete McCoy's 5A basalmedia (Invitrogen Corporation, Carlsbad, Calif.) supplemented with 10%fetal calf serum (Invitrogen Corporation, Carlsbad, Calif.), penicillin100 units per mL, and streptomycin 100 micrograms per mL (InvitrogenCorporation, Carlsbad, Calif.). Cells were routinely passaged bytrypsinization and dilution when they reached 90% confluence. Cells wereseeded into 96-well plates (Falcon-Primaria #353872) at a density of7000 cells/well for use in RT-PCR analysis.

A549 Cells:

The human lung carcinoma cell line A549 was obtained from the AmericanType Culture Collection (ATCC) (Manassas, Va.). A549 cells wereroutinely cultured in DMEM basal media (Invitrogen Corporation,Carlsbad, Calif.) supplemented with 10% fetal calf serum (InvitrogenCorporation, Carlsbad, Calif.), penicillin 100 units per mL, andstreptomycin 100 micrograms per mL (Invitrogen Corporation, Carlsbad,Calif.). Cells were routinely passaged by trypsinization and dilutionwhen they reached 90% confluence.

NHDF Cells:

Human neonatal dermal fibroblast (NHDF) were obtained from the CloneticsCorporation (Walkersville, Md.). NHDFs were routinely maintained inFibroblast Growth Medium (Clonetics Corporation, Walkersville, Md.)supplemented as recommended by the supplier. Cells were maintained forup to 10 passages as recommended by the supplier.

HEK Cells:

Human embryonic keratinocytes (HEK) were obtained from the CloneticsCorporation (Walkersville, Md.). HEKs were routinely maintained inKeratinocyte Growth Medium (Clonetics Corporation, Walkersville, Md.)formulated as recommended by the supplier. Cells were routinelymaintained for up to 10 passages as recommended by the supplier.

HK-2 Cells:

HK-2 (human kidney 2) is a proximal tubular cell (PTC) line derived fromnormal kidney cells immortalized by transduction with human papillomavirus 16 (HPV-16) E6/E7 genes (CRL-2190, American Type CultureCollection, Manassus, Va.). HK-2 cells were routinely cultured inKeratinocyte-Serum Free Medium (17005-042, Invitrogen Corporation,Carlsbad, Calif.) which includes 5 ng/ml recombinant epidermal growthfactor and 0.05 mg/ml bovine pituitary extract. Cells were routinelypassaged by trypsinization and split at a ratio of 1:4 when they reached70-80% confluence. One day prior to transfection, cells were seeded into96-well plates (Falcon-Primaria #353872, BD Biosciences, Bedford, Mass.)at a density of 10,000 cells/well.

b. END Cells:

The mouse brain endothelial cell line b.END was obtained from Dr. WernerRisau at the Max Plank Instititute (Bad Nauheim, Germany). b.END cellswere routinely cultured in DMEM, high glucose (Gibco/Life Technologies,Gaithersburg, Md.) supplemented with 10% fetal calf serum (Gibco/LifeTechnologies, Gaithersburg, Md.). Cells were routinely passaged bytrypsinization and dilution when they reached 90% confluence. Cells wereseeded into 96-well plates (Falcon-Primaria #3872) at a density of 3000cells/well for use in RT-PCR analysis.

Treatment with Antisense Compounds:

When cells reached 65-75% confluency, they were treated witholigonucleotide. For cells grown in 96-well plates, wells were washedonce with 100 μL OPTI-MEM™-1 reduced-serum medium (InvitrogenCorporation, Carlsbad, Calif.) and then treated with 130 μL ofOPTI-MEM™-1 containing 3.75 μg/mL LIPOFECTIN™ (Invitrogen Corporation,Carlsbad, Calif.) and the desired concentration of oligonucleotide.Cells are treated and data are obtained in triplicate. After 4-7 hoursof treatment at 37° C., the medium was replaced with fresh medium. Cellswere harvested 16-24 hours after oligonucleotide treatment.

The concentration of oligonucleotide used varies from cell line to cellline. To determine the optimal oligonucleotide concentration for aparticular cell line, the cells are treated with a positive controloligonucleotide at a range of concentrations. For human cells thepositive control oligonucleotide is selected from either ISIS 13920(TCCGTCATCGCTCCTCAGGG, SEQ ID NO: 1) which is targeted to human H-ras,or ISIS 18078, (GTGCGCGCGAGCCCGAAATC, SEQ ID NO: 2) which is targeted tohuman Jun-N-terminal kinase-2 (JNK2). Both controls are2′-O-methoxyethyl gapmers (2′-O-methoxyethyls shown in bold) with aphosphorothioate backbone. For mouse or rat cells the positive controloligonucleotide is ISIS 15770, ATGCATTCTGCCCCCAAGGA, SEQ ID NO: 3, a2′-O-methoxyethyl gapmer (2′-O-methoxyethyls shown in bold) with aphosphorothioate backbone which is targeted to both mouse and rat c-raf.The concentration of positive control oligonucleotide that results in80% inhibition of c-H-ras (for ISIS 13920), JNK2 (for ISIS 18078) orc-raf (for ISIS 15770) mRNA is then utilized as the screeningconcentration for new oligonucleotides in subsequent experiments forthat cell line. If 80% inhibition is not achieved, the lowestconcentration of positive control oligonucleotide that results in 60%inhibition of c-H-ras, JNK2 or c-raf mRNA is then utilized as theoligonucleotide screening concentration in subsequent experiments forthat cell line. If 60% inhibition is not achieved, that particular cellline is deemed as unsuitable for oligonucleotide transfectionexperiments. The concentrations of antisense oligonucleotides usedherein are from 50 nM to 300 nM.

For Northern blotting or other analysis, cells may be seeded onto 100 mmor other standard tissue culture plates and treated similarly, usingappropriate volumes of medium and oligonucleotide.

Example 10 Analysis of Oligonucleotide Inhibition of SGLT2 Expression

Antisense modulation of SGLT2 expression can be assayed in a variety ofways known in the art. For example, SGLT2 mRNA levels can be quantitatedby, e.g., Northern blot analysis, competitive polymerase chain reaction(PCR), or real-time PCR (RT-PCR). Real-time quantitative PCR ispresently preferred. RNA analysis can be performed on total cellular RNAor poly(A)+ mRNA. The preferred method of RNA analysis of the presentinvention is the use of total cellular RNA as described in otherexamples herein. Methods of RNA isolation are well known in the art.Northern blot analysis is also routine in the art. Real-timequantitative (PCR) can be conveniently accomplished using thecommercially available ABI PRISM™ 7600, 7700, or 7900 Sequence DetectionSystem, available from PE-Applied Biosystems, Foster City, Calif. andused according to manufacturer's instructions.

Protein levels of SGLT2 can be quantitated in a variety of ways wellknown in the art, such as immunoprecipitation, Western blot analysis(immunoblotting), enzyme-linked immunosorbent assay (ELISA) orfluorescence-activated cell sorting (FACS). Antibodies directed to SGLT2can be identified and obtained from a variety of sources, such as theMSRS catalog of antibodies (Aerie Corporation, Birmingham, Mich.), orcan be prepared via conventional monoclonal or polyclonal antibodygeneration methods well known in the art.

Example 11 Design of Phenotypic Assays for the Use of SGLT2 Inhibitors

Phenotypic Assays

Once SGLT2 inhibitors have been identified by the methods disclosedherein, the compounds are further investigated in one or more phenotypicassays, each having measurable endpoints predictive of efficacy in thetreatment of a particular disease state or condition. Phenotypic assays,kits and reagents for their use are well known to those skilled in theart and are herein used to investigate the role and/or association ofSGLT2 in health and disease. Representative phenotypic assays, which canbe purchased from any one of several commercial vendors, include thosefor determining cell viability, cytotoxicity, proliferation or cellsurvival (Molecular Probes, Eugene, Oreg.; PerkinElmer, Boston, Mass.),protein-based assays including enzymatic assays (Panvera, LLC, Madison,Wis.; BD Biosciences, Franklin Lakes, N.J.; Oncogene Research Products,San Diego, Calif.), cell regulation, signal transduction, inflammation,oxidative processes and apoptosis (Assay Designs Inc., Ann Arbor,Mich.), triglyceride accumulation (Sigma-Aldrich, St. Louis, Mo.),angiogenesis assays, tube formation assays, cytokine and hormone assaysand metabolic assays (Chemicon International Inc., Temecula, Calif.;Amersham Biosciences, Piscataway, N.J.).

In one non-limiting example, cells determined to be appropriate for aparticular phenotypic assay (i.e., MCF-7 cells selected for breastcancer studies; adipocytes for obesity studies) are treated with SGLT2inhibitors identified from the in vitro studies as well as controlcompounds at optimal concentrations which are determined by the methodsdescribed above. At the end of the treatment period, treated anduntreated cells are analyzed by one or more methods specific for theassay to determine phenotypic outcomes and endpoints.

Phenotypic endpoints include changes in cell morphology over time ortreatment dose as well as changes in levels of cellular components suchas proteins, lipids, nucleic acids, hormones, saccharides or metals.Measurements of cellular status which include pH, stage of the cellcycle, intake or excretion of biological indicators by the cell, arealso endpoints of interest.

Analysis of the genotype of the cell (measurement of the expression ofone or more of the genes of the cell) after treatment is also used as anindicator of the efficacy or potency of the SGLT2 inhibitors. Hallmarkgenes, or those genes suspected to be associated with a specific diseasestate, condition, or phenotype, are measured in both treated anduntreated cells.

Example 12 RNA Isolation

Poly(A)+ mRNA Isolation

Poly(A)+ mRNA was isolated according to Miura et al., (Clin. Chem.,1996, 42, 1758-1764). Other methods for poly(A)+ mRNA isolation areroutine in the art. Briefly, for cells grown on 96-well plates, growthmedium was removed from the cells and each well was washed with 200 μLcold PBS. 60 μL lysis buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 MNaCl, 0.5% NP-40, 20 mM vanadyl-ribonucleoside complex) was added toeach well, the plate was gently agitated and then incubated at roomtemperature for five minutes. 55 μL of lysate was transferred to Oligod(T) coated 96-well plates (AGCT Inc., Irvine Calif.). Plates wereincubated for 60 minutes at room temperature, washed 3 times with 200 μLof wash buffer (10 mM Tris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl). After thefinal wash, the plate was blotted on paper towels to remove excess washbuffer and then air-dried for 5 minutes. 60 μL of elution buffer (5 mMTris-HCl pH 7.6), preheated to 70° C., was added to each well, the platewas incubated on a 90° C. hot plate for 5 minutes, and the eluate wasthen transferred to a fresh 96-well plate.

Cells grown on 100 mm or other standard plates may be treated similarly,using appropriate volumes of all solutions.

Total RNA Isolation

Total RNA was isolated using an RNEASY 96™ kit and buffers purchasedfrom Qiagen Inc. (Valencia, Calif.) following the manufacturer'srecommended procedures. Briefly, for cells grown on 96-well plates,growth medium was removed from the cells and each well was washed with200 μL cold PBS. 150 μL Buffer RLT was added to each well and the platevigorously agitated for 20 seconds. 150 μL of 70% ethanol was then addedto each well and the contents mixed by pipetting three times up anddown. The samples were then transferred to the RNEASY 96™ well plateattached to a QIAVAC™ manifold fitted with a waste collection tray andattached to a vacuum source. Vacuum was applied for 1 minute. 500 μL ofBuffer RW1 was added to each well of the RNEASY 96™ plate and incubatedfor 15 minutes and the vacuum was again applied for 1 minute. Anadditional 500 μL of Buffer RW1 was added to each well of the RNEASY 96™plate and the vacuum was applied for 2 minutes. 1 mL of Buffer RPE wasthen added to each well of the RNEASY 96™ plate and the vacuum appliedfor a period of 90 seconds. The Buffer RPE wash was then repeated andthe vacuum was applied for an additional 3 minutes. The plate was thenremoved from the QIAVAC™ manifold and blotted dry on paper towels. Theplate was then re-attached to the QIAVAC™ manifold fitted with acollection tube rack containing 1.2 mL collection tubes. RNA was theneluted by pipetting 140 μL of RNAse free water into each well,incubating 1 minute, and then applying the vacuum for 3 minutes.

The repetitive pipetting and elution steps may be automated using aQIAGEN Bio-Robot 9604 (Qiagen, Inc., Valencia Calif.). Essentially,after lysing of the cells on the culture plate, the plate is transferredto the robot deck where the pipetting, DNase treatment and elution stepsare carried out.

Example 13 Real-Time Quantitative PCR Analysis of SGLT2 mRNA Levels

Quantitation of SGLT2 mRNA levels was accomplished by real-timequantitative PCR using the ABI PRISM™ 7600, 7700, or 7900 SequenceDetection System (PE-Applied Biosystems, Foster City, Calif.) accordingto manufacturer's instructions. This is a closed-tube, non-gel-based,fluorescence detection system which allows high-throughput quantitationof polymerase chain reaction (PCR) products in real-time. As opposed tostandard PCR in which amplification products are quantitated after thePCR is completed, products in real-time quantitative PCR are quantitatedas they accumulate. This is accomplished by including in the PCRreaction an oligonucleotide probe that anneals specifically between theforward and reverse PCR primers, and contains two fluorescent dyes. Areporter dye (e.g., FAM or JOE, obtained from either PE-AppliedBiosystems, Foster City, Calif., Operon Technologies Inc., Alameda,Calif. or Integrated DNA Technologies Inc., Coralville, Iowa) isattached to the 5′ end of the probe and a quencher dye (e.g., TAMRA,obtained from either PE-Applied Biosystems, Foster City, Calif., OperonTechnologies Inc., Alameda, Calif. or Integrated DNA Technologies Inc.,Coralville, Iowa) is attached to the 3′ end of the probe. When the probeand dyes are intact, reporter dye emission is quenched by the proximityof the 3′ quencher dye. During amplification, annealing of the probe tothe target sequence creates a substrate that can be cleaved by the5′-exonuclease activity of Taq polymerase. During the extension phase ofthe PCR amplification cycle, cleavage of the probe by Taq polymerasereleases the reporter dye from the remainder of the probe (and hencefrom the quencher moiety) and a sequence-specific fluorescent signal isgenerated. With each cycle, additional reporter dye molecules arecleaved from their respective probes, and the fluorescence intensity ismonitored at regular intervals by laser optics built into the ABI PRISM™Sequence Detection System. In each assay, a series of parallel reactionscontaining serial dilutions of mRNA from untreated control samplesgenerates a standard curve that is used to quantitate the percentinhibition after antisense oligonucleotide treatment of test samples.

Prior to quantitative PCR analysis, primer-probe sets specific to thetarget gene being measured are evaluated for their ability to be“multiplexed” with a GAPDH amplification reaction. In multiplexing, boththe target gene and the internal standard gene GAPDH are amplifiedconcurrently in a single sample. In this analysis, mRNA isolated fromuntreated cells is serially diluted. Each dilution is amplified in thepresence of primer-probe sets specific for GAPDH only, target gene only(“single-plexing”), or both (multiplexing). Following PCR amplification,standard curves of GAPDH and target mRNA signal as a function ofdilution are generated from both the single-plexed and multiplexedsamples. If both the slope and correlation coefficient of the GAPDH andtarget signals generated from the multiplexed samples fall within 10% oftheir corresponding values generated from the single-plexed samples, theprimer-probe set specific for that target is deemed multiplexable. Othermethods of PCR are also known in the art.

PCR reagents were obtained from Invitrogen Corporation, (Carlsbad,Calif.). RT-PCR reactions were carried out by adding 20 μL PCR cocktail(2.5× PCR buffer minus MgCl₂, 6.6 mM MgCl₂, 375 μM each of dATP, dCTP,dCTP and dGTP, 375 nM each of forward primer and reverse primer, 125 nMof probe, 4 Units RNAse inhibitor, 1.25 Units PLATINUM® Taq, 5 UnitsMuLV reverse transcriptase, and 2.5× ROX dye) to 96-well platescontaining 30 μL total RNA solution (20-200 ng). The RT reaction wascarried out by incubation for 30 minutes at 48° C. Following a 10 minuteincubation at 95° C. to activate the PLATINUM® Taq, 40 cycles of atwo-step PCR protocol were carried out: 95° C. for 15 seconds(denaturation) followed by 60° C. for 1.5 minutes (annealing/extension).

Gene target quantities obtained by real time RT-PCR are normalized usingeither the expression level of GAPDH, a gene whose expression isconstant, or by quantifying total RNA using RiboGreen™ (MolecularProbes, Inc. Eugene, Oreg.). GAPDH expression is quantified by real timeRT-PCR, by being run simultaneously with the target, multiplexing, orseparately. Total RNA is quantified using RiboGreen™ RNA quantificationreagent (Molecular Probes, Inc. Eugene, Oreg.). Methods of RNAquantification by RiboGreen™ are taught in Jones, L. J., et al,(Analytical Biochemistry, 1998, 265, 368-374).

In this assay, 170 μL of RiboGreen™ working reagent (RiboGreen™ reagentdiluted 1:350 in 10 mM Tris-HCl, 1 mM EDTA, pH 7.5) is pipetted into a96-well plate containing 30 μL purified, cellular RNA. The plate is readin a CytoFluor 4000 (PE Applied Biosystems) with excitation at 485 nmand emission at 530 nm.

Probes and primers to human SGLT2 were designed to hybridize to a humanSGLT2 sequence, using published sequence information (GenBank accessionnumber NM_(—)003041.1, incorporated herein as SEQ ID NO: 4). For humanSGLT2 the PCR primers were: forward primer: TCGGCGTGCCCAGCT (SEQ ID NO:5) reverse primer: AGAACAGCACAATGGCGAAGT (SEQ ID NO: 6) and the PCRprobe was: FAM-TCCTCTGCGGCGTGCACTACCTC-TAMRA (SEQ ID NO: 7)

where FAM is the fluorescent dye and TAMRA is the quencher dye. Forhuman GAPDH the PCR primers were: forward primer: GAAGGTGAAGGTCGGAGTC(SEQ ID NO: 8) reverse primer: GAAGATGGTGATGGGATTTC (SEQ ID NO: 9) andthe PCR probe was: 5′ JOE-CAAGCTTCCCGTTCTCAGCC- (SEQ ID NO: 10) TAMRA 3′where JOE is the fluorescent reporter dye and TAMRA is the quencher dye.

Probes and primers to mouse SGLT2 were designed to hybridize to a mouseSGLT2 sequence, using published sequence information (the concatenationof the sequences with the GenBank accession numbers: AJ292928, AW106808,AI789450, AW046901, the complement of AI647605, the complement ofAW107250, and the complement of AI788744, incorporated herein as SEQ IDNO: 11). For mouse SGLT2 the PCR primers were: forward primer:TGTTGGACCCTCACAAAGAGTAAG (SEQ ID NO: 12) reverse primer:GCTGTATTCTTGCCCTGTTCCT (SEQ ID NO: 13) and the PCR probe was:FAM-TTCTGGGATCCACTCCAAGCTGCTCA- (SEQ ID NO: 14) TAMRA

where FAM is the fluorescent reporter dye and TAMRA is the quencher dye.For mouse GAPDH the PCR primers were: forward primer:GGCAAATTCAACGGCACAGT (SEQ ID NO: 15) reverse primer:GGGTCTCGCTCCTGGAAGAT (SEQ ID NO: 16) and the PCR probe was: 5′ JOE- (SEQID NO: 17) AAGGCCGAGAATGGGAAGCTTGTCATC- TAMRA 3′where JOE is the fluorescent reporter dye and TAMRA is the quencher dye.

Example 14 Northern Blot Analysis of SGLT2 mRNA Levels

Eighteen hours after antisense treatment, cell monolayers were washedtwice with cold PBS and lysed in 1 mL RNAZOL™ (TEL-TEST “B” Inc.,Friendswood, Tex.). Total RNA was prepared following manufacturer'srecommended protocols. Twenty micrograms of total RNA was fractionatedby electrophoresis through 1.2% agarose gels containing 1.1%formaldehyde using a MOPS buffer system (AMRESCO, Inc. Solon, Ohio). RNAwas transferred from the gel to HYBOND™-N+ nylon membranes (AmershamPharmacia Biotech, Piscataway, N.J.) by overnight capillary transferusing a Northern/Southern Transfer buffer system (TEL-TEST “B” Inc.,Friendswood, Tex.). RNA transfer was confirmed by UV visualization.Membranes were fixed by UV cross-linking using a STRATALINKER™ UVCrosslinker 2400 (Stratagene, Inc, La Jolla, Calif.) and then probedusing QUICKHYB™ hybridization solution (Stratagene, La Jolla, Calif.)using manufacturer's recommendations for stringent conditions.

To detect human SGLT2, a human SGLT2 specific probe was prepared by PCRusing the forward primer TCGGCGTGCCCAGCT (SEQ ID NO: 5) and the reverseprimer AGAACAGCACAATGGCGAAGT (SEQ ID NO: 6). To normalize for variationsin loading and transfer efficiency membranes were stripped and probedfor human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA(Clontech, Palo Alto, Calif.).

To detect mouse SGLT2, a mouse SGLT2 specific probe was prepared by PCRusing the forward primer TGTTGGACCCTCACAAAGAGTAAG (SEQ ID NO: 12) andthe reverse primer GCTGTATTCTTGCCCTGTTCCT (SEQ ID NO: 13). To normalizefor variations in loading and transfer efficiency membranes werestripped and probed for mouse glyceraldehyde-3-phosphate dehydrogenase(GAPDH) RNA (Clontech, Palo Alto, Calif.).

Hybridized membranes were visualized and quantitated using aPHOSPHORIMAGER™ and IMAGEQUANT™ Software V3.3 (Molecular Dynamics,Sunnyvale, Calif.). Data was normalized to GAPDH levels in untreatedcontrols.

Example 15 Antisense Inhibition of Human SGLT2 Expression by ChimericPhosphorothioate Oligonucleotides Having 2′-MOE Wings and a Deoxy Gap

In accordance with the present invention, a series of antisensecompounds was designed to target different regions of the human SGLT2RNA, using published sequences (GenBank accession number NM_(—)003041.1,incorporated herein as SEQ ID NO: 4). The compounds are shown inTable 1. “Target site” indicates the first (5′-most) nucleotide numberon the particular target sequence to which the compound binds. Allcompounds in Table 1 are chimeric oligonucleotides (“gapmers”) 20nucleotides in length, composed of a central “gap” region consisting often 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′directions) by five-nucleotide “wings”. The wings are composed of2′-methoxyethyl (2′-MOE)nucleotides. The internucleoside (backbone)linkages are phosphorothioate (P═S) throughout the oligonucleotide. Allcytidine residues are 5-methylcytidines. The compounds were analyzed fortheir effect on human SGLT2 mRNA levels by quantitative real-time PCR asdescribed in other examples herein. HK-2 cells were treated with 500 nMof antisense oligonucleotide mixed with 15 μg/mL LIPOFECTIN. Data areaverages from three experiments in which HK-2 cells were treated withthe antisense oligonucleotides of the present invention. If present,“N.D.” indicates “no data”. TABLE 1 Inhibition of human SGLT2 mRNAlevels by chimeric phosphorothioate oligonucleotides having 2′-MOE wingsand a deoxy gap TARGET SEQ ID TARGET % SEQ ID ISIS # REGION NO SITESEQUENCE INHIB NO 337873 Start Codon 4 1 tctccccaggatctgccccc 17 18337874 Start Codon 4 15 gtgtgctcctccattctccc 41 19 337875 Coding 4 42cccatctctggtgccgagcc 33 20 337876 Coding 4 70 aggattgtcaatcagggcct 49 21337877 Coding 4 95 atgcagcaatgactaggatg 45 22 337878 Coding 4 124caagccaacgccaatgacca 54 23 337879 Coding 4 150 cctctgttggttctgcacat 2624 337880 Coding 4 182 tgcgtcctgccaggaagtag 25 25 337881 Coding 4 204ccaaccggccaccacaccat 45 26 337882 Coding 4 262 agtccctgccaggcccacaa 4327 337883 Coding 4 291 ccagcaacagccaagccact 37 28 337884 Coding 4 354aggtacacgggtgcaaacag 48 29 337885 Coding 4 384 tactgtggcatcgtgatgac 2830 337886 Coding 4 426 aggtagaggcggatgcggcg 24 31 337887 Coding 4 442aagggagagcacagacaggt 50 32 337888 Coding 4 474 tccactgagatcttggtgaa 4133 337889 Coding 4 501 tggatgaatacagctccgga 49 34 337890 Coding 4 529ggcatagatgttccagccca 36 35 337891 Coding 4 560 tcatggtgatgcccagaagc 2336 337892 Coding 4 577 tcctgtcaccgtgtaaatca 39 37 337893 Coding 4 600gtgtacatcagcgcggccag 33 38 337894 Coding 4 624 atgacgaaggtctgtaccgt 4139 337895 Coding 4 651 cccatgaggatgcaggcgcc 55 40 337896 Coding 4 694gtcgaagagacccgaatacc 30 41 337897 Coding 4 716 aagtcgctgctcccaggtat 0 42337898 Coding 4 772 tcgatagcagaagctggaga 47 43 337899 Coding 4 849agtccgaggagcagcgcggg 5 44 337900 Coding 4 884 ggtcgctgcaccagtaccag 29 45337901 Coding 4 909 gccaggcagcgctgcacgat 22 46 337902 Coding 4 944tgcagcccgccttgatgtgg 67 47 337903 Coding 4 954 ccacacaggatgcagcccgc 3748 337904 Coding 4 991 catgaccatgagaaacatgg 43 49 337905 Coding 4 1006gctgatcatgcctggcatga 54 50 337906 Coding 4 1033 cgccacctcgtctgggtaca 4551 337907 Coding 4 1051 cacctcaggcaccacgcacg 54 52 337908 Coding 4 1073ccgtgccgcacacgcgcctg 34 53 337909 Coding 4 1100 ggtaggcgatgttggagcag 3054 337910 Coding 4 1122 atgagcttcacgacgagccg 48 55 337911 Coding 4 1151ccagcatgagtccgcgcaga 50 56 337912 Coding 4 1180 cgaggacatgagcgcggcca 7157 337913 Coding 4 1211 gcgtgctgctgctgttgaag 37 58 337914 Coding 4 1232tgtagatgtccatggtgaag 39 59 337915 Coding 4 1272 agcagcagctcgcggtcgcc 2160 337916 Coding 4 1292 ccacccagagccgtcccacc 47 61 337917 Coding 4 1319aggccaccgacactaccacg 38 62 337918 Coding 4 1360 gaagagctgcccgccctgtg 3863 337919 Coding 4 1372 ctggatgtaatcgaagagct 45 64 337920 Coding 4 1415cgaagacggcggacacgggc 3 65 337921 Coding 4 1433 gcacgaagagcgccagcacg 3266 337922 Coding 4 1453 gccctgctcattaacgcgcg 34 67 337923 Coding 4 1479aggcccccgatgagtcccca 48 68 337924 Coding 4 1497 cgtgccaggcccatcagcag 3769 337925 Coding 4 1526 ccgagccgaaggagaactcg 47 70 337926 Coding 4 1544agggctgcacacagctgccc 0 71 337927 Coding 4 1570 gccgcagaggaaagctgggc 1572 337928 Coding 4 1595 caatggcgaagtagaggtag 37 73 337929 Coding 4 1615gccagagcagaagaacagca 41 74 337930 Coding 4 1641 cacagggagaccgtgagggt 1175 337931 Coding 4 1677 aggcggtggaggtgctttct 29 76 337932 Coding 4 1706cctccttgctatgccggaga 47 77 337933 Coding 4 1729 atcagcatccaggtcctccc 078 337934 Coding 4 1763 cattctgtacagggagtgag 50 79 337935 Coding 4 1788atctccatggcactctctgg 58 80 337936 Coding 4 1835 gcaggcactggcggaagagg 2981 337937 Coding 4 1861 acctctgctcattccacaaa 56 82 337938 Coding 4 1881ggcggaggactgcccacccc 22 83 337939 Coding 4 1917 cgcctggctgctgccgctgc 1184 337940 Coding 4 1939 gtcctcgctgatgtcctcca 40 85 337941 Coding 4 1972ggcattgaggttgaccacac 2 86 337942 Coding 4 2003 agaggaacacggccactgcc 8 87337943 Coding 4 2014 atagaagccccagaggaaca 39 88 337944 Stop Codon 4 2025tggtcttaggcatagaagcc 28 89 337945 3′UTR 4 2048 tggcttatggtgtccaacgc 3590 337946 3′UTR 4 2072 tcacccccacttcctgtgag 42 91 337947 3′UTR 4 2120tctcaccccactgccccttc 38 92 337948 3′UTR 4 2158 caggcagaggaaggccggga 3893 337949 3′UTR 4 2197 cctcatgggaagtgactgcc 37 94 337950 3′UTR 4 2230ttccttagggcaactgcagc 34 95

As shown in Table 1, SEQ ID NOs 19, 20, 21, 22, 23, 26, 27, 28, 29, 32,33, 34, 35, 37, 38, 39, 40, 41, 43, 47, 48, 49, 50, 51, 52, 53, 54, 55,56, 57, 58, 59, 61, 62, 63, 64, 66, 67, 68, 69, 70, 73, 74, 77, 79, 80,82, 85, 88, 90, 91, 92, 93, 94 and 95 demonstrated at least 30%inhibition of human SGLT2 expression in this assay. The target regionsto which these sequences are complementary are herein referred to as“suitable target segments” and are therefor suitable for targeting bycompounds of the present invention. These target segments are shown inTable 3. These sequences are shown to contain thymine (T) but one ofskill in the art will appreciate that thymine (T) is generally replacedby uracil (U) in RNA sequences. The sequences represent the reversecomplement of the suitable antisense compounds shown in Table 1, “Targetsite” indicates the first (5′-most) nucleotide number on the particulartarget nucleic acid to which the oligonucleotide binds. Also shown inTable 3 is the species in which each of the suitable target segments wasfound.

Example 16 Antisense Inhibition of Mouse SGLT2 Expression by ChimericPhoshorothioate Oligonucleotides Having 2′-MOE Wings and a Deoxy Gap

In accordance with the present invention, a second series of antisensecompounds was designed to target different regions of the mouse SGLT2RNA, using published sequences (the concatenation of the sequences withthe GenBank accession numbers: AJ292928, AW106808, AI789450, AW046901,the complement of AI647605, the complement of AW107250, and thecomplement of AI788744, incorporated herein as SEQ ID NO: 11; GenBankaccession number AJ292928. 1, incorporated herein as SEQ ID NO: 96; andGenBank accession number AW045170.1, incorporated herein as SEQ ID NO:97). The compounds are shown in Table 2. “Target site” indicates thefirst (5′-most) nucleotide number on the particular target nucleic acidto which the compound binds. All compounds in Table 2 are chimericoligonucleotides (“gapmers”) 20 nucleotides in length, composed of acentral “gap” region consisting of ten 2′-deoxynucleotides, which isflanked on both sides (5′ and 3′ directions) by five-nucleotide “wings”.The wings are composed of 2′-methoxyethyl (2′-MOE) nucleotides. Theinternucleoside (backbone) linkages are phosphorothioate (P═S)throughout the oligonucleotide. All cytidine residues are5-methylcytidines. The compounds were analyzed for their effect on mouseSGLT2 mRNA levels by quantitative real-time PCR as described in otherexamples herein. Data are averages from three experiments in which b.ENDcells were treated with 100 nM of the antisense oligonucleotides of thepresent invention. The positive control for each datapoint is identifiedin the table by sequence ID number. If present, “N.D.” indicates “nodata”. TABLE 2 Inhibition of mouse SGLT2 mRNA levels by chimericphosphorothioate oligonucleotides having 2′-MOE wings and a deoxy gapTARGET CONTROL SEQ ID TARGET % SEQ ID SEQ ID ISIS # REGION NO SITESEQUENCE INHIB NO NO 145725 Coding 11 27 tgctccccaagttcagagcc 16 98 1145726 Coding 11 39 atcaggaccttctgctcccc 30 99 1 145727 Coding 11 50caggattatcaatcaggacc 15 100 1 145728 Coding 11 62 ccagaatgtcagcaggatta 9101 1 145729 Coding 11 93 ccaatgaccagcaggaaata 15 102 1 145730 Coding 11117 ctgaacatagaccacaagcc 0 103 1 145731 Coding 11 127tctattggttctgaacatag 9 104 1 145732 Coding 11 138 ccaactgtgcctctattggt43 105 1 145733 Coding 11 148 gaagtagccaccaactgtgc 16 106 1 145734Coding 11 189 gaggctccaaccggccacca 43 107 1 145735 Coding 11 213ctgccgatgttgctggcgaa 2 108 1 145736 Coding 11 230 ggcccacaaaatgaccgctg44 109 1 145737 Coding 11 261 gccaagccacttgctgcacc 29 110 1 145738Coding 11 294 acgaagagcgcattccactc 7 111 1 145739 Coding 11 299gcaccacgaagagcgcattc 0 112 1 145740 Coding 11 375 cgcttgcggaggtactgagg 0113 1 145741 Coding 11 420 agcgagagcacggacaggta 0 114 1 145742 Coding 11462 gagaacatatccaccgagat 5 115 1 145743 Coding 11 490cagggcctgttgaatgaata 0 116 1 145744 Coding 11 550 cacagtataaatcatggtga35 117 1 145745 Coding 11 581 ctgtgtacatcagtgccgcc 18 118 1 145746Coding 11 592 ctgcacagtgtctgtgtaca 7 119 1 145747 Coding 11 605gaatgacgaaggtctgcaca 14 120 1 145748 Coding 11 616 ggccccggcaagaatgacga25 121 1 145749 Coding 11 659 agtacccgcccacttcatgg 0 122 1 145750 Coding11 706 acccgtcagtgaagtcattg 18 123 1 145751 Coding 11 784gtcacgcagcaggtgatagg 24 124 1 145752 Coding 11 795 cctgtcacagggtcacgcag40 125 1 145753 Coding 11 840 gagacaatggtaagccccag 20 126 1 145754Coding 11 902 tcagattctttccagccagg 12 127 1 145755 Coding 11 912ttgatgtgagtcagattctt 13 128 1 145756 Coding 11 998 ggtagagaatgcggctgatc8 129 1 145757 Coding 11 1039 ccgcttacacacctcaggta 32 130 1 145758Coding 11 1050 gtgccacacacccgcttaca 39 131 1 145759 Coding 11 1068ttagagcagcccacctcagt 28 132 1 145760 Coding 11 1081 tgggtaggcgatgttagagc15 133 1 145761 Coding 11 1113 agaccattgggcatgagctt 2 134 1 145762Coding 11 1128 agcatgagtccgcgcagacc 0 135 1 145763 Coding 11 1142ccagcatgactgccagcatg 22 136 1 145764 Coding 11 1177 gttaaagatggatgccagag0 137 1 145765 Coding 11 1246 cagctccttatcacctgcac 55 138 1 145766Coding 11 1320 gctgcctgcaccactggcag 44 139 1 145767 Coding 11 1393aaagaccgcagacacttgag 0 140 1 145768 Coding 11 1403 gtgcaagcacaaagaccgca6 141 1 145769 Coding 11 1475 gagctaggcccatcagcagg 55 142 1 145770Coding 11 1485 ggtatgagacgagctaggcc 0 143 1 145771 Coding 11 1496agaagaactcgggtatgaga 0 144 1 145772 Coding 11 1524 gagggtcgcacacagctgcc8 145 1 145773 Coding 11 1563 tagaggtagtgtacccgaca 0 146 1 145774 Coding11 1682 ccttgctgtgccggagactg 40 147 1 145775 Coding 11 1707tcagcatccaggtcctcccg 46 148 1 145776 Coding 11 1722 ggaccttctaactcatcagc2 149 1 145777 Coding 11 1765 cattgcacattcctggcccc 23 150 1 145778Coding 11 1839 ttgctcatcccacagaacca 15 151 1 145779 Coding 11 1851cctgacccactcttgctcat 1 152 1 145780 Coding 11 1881 gccacctcctcggtagtggg21 153 1 145781 Coding 11 1909 gatgtcctccagccgcctgg 0 154 1 145782Coding 11 1921 gggatcctcactgatgtcct 25 155 1 145783 Coding 11 1953agggcattgaggttgactac 11 156 1 145784 Coding 11 1992 tagaagccccagaggaacac0 157 1 145785 3′UTR 11 2164 aatcaaatggactggacccc 0 158 1 145786 3′UTR11 2174 agtgacaaccaatcaaatgg 10 159 1 145787 3′UTR 11 2186catcttgtgggaagtgacaa 14 160 1 145788 3′UTR 11 2199 accaattggccatcatcttg0 161 1 145789 3′UTR 11 2237 ggagggcagttttatttttg 20 162 1 145790exon:intron 96 2123 caatgtctcacccacaagcc 4 163 1 145791 intron 96 2239ctaaatctaggtttctccct 11 164 1 145792 intron 96 2291 ttttgcacaatccagaaggt9 165 1 145793 intron 96 2407 gaccttaaatataggctgct 0 166 1 145794 intron96 2477 aacccaggccctaatcctag 4 167 1 145795 intron 96 2551aggctgaagattaaccagcc 8 168 1 145796 intron 96 2595 ttggacttccttagcttcct9 169 1 145797 exon:intron 96 2647 gaacatagactgggaaacag 0 170 1 145798intron 96 2797 gaggctccaacctgggtggc 12 171 1 145799 intron 97 133tccagcaaatgaacctgtgt 0 172 1 145800 intron 97 284 cacagcggaagtgcctgggc21 173 1 145801 intron 97 316 tgtcctagtcctcacaccca 12 174 1 145802intron 97 338 gggacagcatcctgagcagg 25 175 1

As shown in Table 2, SEQ ID NOs 99, 105, 107, 109, 110, 117, 121, 124,125, 126, 130, 131, 132, 136, 138, 139, 142, 147, 148, 150, 153, 155,162, 173 and 175 demonstrated at least 20% inhibition of mouse SGLT2expression in this experiment. Also suitable are SEQ ID NOs 105, 119 and135. The target regions to which these sequences are complementary areherein referred to as “suitable target segments” and are thereforesuitable for targeting by compounds of the present invention. Thesetarget segments are shown in Table 3. These sequences are shown tocontain thymine (T) but one of skill in the art will appreciate thatthymine (T) is generally replaced by uracil (U) in RNA sequences. Thesequences represent the reverse complement of the preferred antisensecompounds shown in Tables 1 and 2. “Target site” indicates the first(5′-most) nucleotide number on the particular target nucleic acid towhich the oligonucleotide binds. Also shown in Table 3 is the species inwhich each of the suitable target segments was found. TABLE 3 Sequenceand position of preferred target segments identified in human and mouseSGLT2. TARGET REV SITE SEQ ID TARGET COMP OF ACTIVE SEQ ID ID NO SITESEQUENCE SEQ ID IN NO 253571 4 15 gggagaatggaggagcacac 19 H. sapiens 176253572 4 42 ggctcggcaccagagatggg 20 H. sapiens 177 253573 4 70aggccctgattgacaatcct 21 H. sapiens 178 253574 4 95 catcctagtcattgctgcat22 H. sapiens 179 253575 4 124 tggtcattggcgttggcttg 23 H. sapiens 180253578 4 204 atggtgtggtggccggttgg 26 H. sapiens 181 253579 4 262ttgtgggcctggcagggact 27 H. sapiens 182 253580 4 291 agtggcttggctgttgctgg28 H. sapiens 183 253581 4 354 ctgtttgcacccgtgtacct 29 H. sapiens 184253584 4 442 acctgtctgtgctctccctt 32 H. sapiens 185 253585 4 474ttcaccaagatctcagtgga 33 H. sapiens 186 253586 4 501 tccggagctgtattcatcca34 H. sapiens 187 253587 4 529 tgggctggaacatctatgcc 35 H. sapiens 188253589 4 577 tgatttacacggtgacagga 37 H. sapiens 189 253590 4 600ctggccgcgctgatgtacac 38 H. sapiens 190 253591 4 624 acggtacagaccttcgtcat39 H. sapiens 191 253592 4 651 ggcgcctgcatcctcatggg 40 H. sapiens 192253593 4 694 ggtattcgggtctcttcgac 41 H. sapiens 193 253595 4 772tctccagcttctgctatcga 43 H. sapiens 194 253599 4 944 ccacatcaaggcgggctgca47 H. sapiens 195 253600 4 954 gcgggctgcatcctgtgtgg 48 H. sapiens 196253601 4 991 ccatgtttctcatggtcatg 49 H. sapiens 197 253602 4 1006tcatgccaggcatgatcagc 50 H. sapiens 198 253603 4 1033tgtacccagacgaggtggcg 51 H. sapiens 199 253604 4 1051cgtgcgtggtgcctgaggtg 52 H. sapiens 200 253605 4 1073caggcgcgtgtgcggcacgg 53 H. sapiens 201 253606 4 1100ctgctccaacatcgcctacc 54 H. sapiens 202 253607 4 1122cggctcgtcgtgaagctcat 55 H. sapiens 203 253608 4 1151tctgcgcggactcatgctgg 56 H. sapiens 204 253609 4 1180tggccgcgctcatgtcctcg 57 H. sapiens 205 253610 4 1211cttcaacagcagcagcacgc 58 H. sapiens 206 253611 4 1232cttcaccatggacatctaca 59 H. sapiens 207 253613 4 1292ggtgggacggctctgggtgg 61 H. sapiens 208 253614 4 1319cgtggtagtgtcggtggcct 62 H. sapiens 209 253615 4 1360cacagggcgggcagctcttc 63 H. sapiens 210 253616 4 1372agctcttcgattacatccag 64 H. sapiens 211 253618 4 1433cgtgctggcgctcttcgtgc 66 H. sapiens 212 253619 4 1453cgcgcgttaatgagcagggc 67 H. sapiens 213 253620 4 1479tggggactcatcgggggcct 68 H. sapiens 214 253621 4 1497ctgctgatgggcctggcacg 69 H. sapiens 215 253622 4 1526cgagttctccttcggctcgg 70 H. sapiens 216 253625 4 1595ctacctctacttcgccattg 73 H. sapiens 217 253626 4 1615tgctgttcttctgctctggc 74 H. sapiens 218 253629 4 1706tctccggcatagcaaggagg 77 H. sapiens 219 253631 4 1763ctcactccctgtacagaatg 79 H. sapiens 220 253632 4 1788ccagagagtgccatggagat 80 H. sapiens 221 253634 4 1861tttgtggaatgagcagaggt 82 H. sapiens 222 253637 4 1939tggaggacatcagcgaggac 85 H. sapiens 223 253640 4 2014tgttcctctggggcttctat 88 H. sapiens 224 253642 4 2048gcgttggacaccataagcca 90 H. sapiens 225 253643 4 2072ctcacaggaagtgggggtga 91 H. sapiens 226 253644 4 2120gaaggggcagtggggtgaga 92 H. sapiens 227 253645 4 2158tcccggccttcctctgcctg 93 H. sapiens 228 253646 4 2197ggcagtcacttcccatgagg 94 H. sapiens 229 253647 4 2230gctgcagttgccctaaggaa 95 H. sapiens 230 58683 11 39 ggggagcagaaggtcctgat99 M. musculus 231 58689 11 138 accaatagaggcacagttgg 105 M. musculus 23258691 11 189 tggtggccggttggagcctc 107 M. musculus 233 58693 11 230cagcggtcattttgtgggcc 109 M. musculus 234 58694 11 261ggtgcagcaagtggcttggc 110 M. musculus 235 58701 11 550tcaccatgatttatactgtg 117 M. musculus 236 58705 11 616tcgtcattcttgccggggcc 121 M. musculus 237 58708 11 784cctatcacctgctgcgtgac 124 M. musculus 238 58709 11 795ctgcgtgaccctgtgacagg 125 M. musculus 239 58710 11 840ctggggcttaccattgtctc 126 M. musculus 240 58714 11 1039tacctgaggtgtgtaagcgg 130 M. musculus 241 58715 11 1050tgtaagcgggtgtgtggcac 131 M. musculus 242 58716 11 1068actgaggtgggctgctctaa 132 M. musculus 243 58720 11 1142catgctggcagtcatgctgg 136 M. musculus 244 58722 11 1246gtgcaggtgataaggagctg 138 M. musculus 245 58723 11 1320ctgccagtggtgcaggcagc 139 M. musculus 246 58726 11 1475cctgctgatgggcctagctc 142 M. musculus 247 58731 11 1682cagtctccggcacagcaagg 147 M. musculus 248 58732 11 1707cgggaggacctggatgctga 148 M. musculus 249 58734 11 1765ggggccaggaatgtgcaatg 150 M. musculus 250 58737 11 1881cccactaccgaggaggtggc 153 M. musculus 251 58739 11 1921aggacatcagtgaggatccc 155 M. musculus 252 58746 11 2237caaaaataaaactgccctcc 162 M. musculus 253 58757 97 284gcccaggcacttccgctgtg 173 M. musculus 254 58759 97 338cctgctcaggatgctgtccc 175 M. musculus 255

As these “suitable target segments” have been found by experimentationto be open to, and accessible for, hybridization with the antisensecompounds of the present invention, one of skill in the art willrecognize or be able to ascertain, using no more than routineexperimentation, further embodiments of the invention that encompassother compounds that specifically hybridize to these suitable targetsegments and consequently inhibit the expression of SGLT2.

According to the present invention, antisense compounds includeantisense oligomeric compounds, antisense oligonucteotides, ribozymes,external guide sequence (EGS) oligonucleotides, alternate splicers, andother short oligomeric compounds which hybridize to at least a portionof the target nucleic acid.

Example 17 Western Blot Analysis of SGLT2 Protein Levels

Western blot analysis (immunoblot analysis) is carried out usingstandard methods. Cells are harvested 16-20 h after oligonucleotidetreatment, washed once with PBS, suspended in Laemmli buffer (100μl/well), boiled for 5 minutes and loaded on a 16% SDS-PAGE gel. Gelsare run for 1.5 hours at 150 V, and transferred to membrane for westernblotting. Appropriate primary antibody directed to SGLT2 is used, with aradiolabeled or fluorescently labeled secondary antibody directedagainst the primary antibody species. Bands are visualized using aPHOSPHORIMAGER™ (Molecular Dynamics, Sunnyvale Calif.).

Example 18 Design of Chemically Modified Antisense Compounds TargetingSGLT2

A series of chemically modified antisense compounds were designed usingthe sequence of ISIS 145733 (SEQ ID NO: 106), ISIS 145742 (SEQ ID NO:265) or ISIS 145746 (SEQ ID NO: 266). Modifications were made to theinternucleoside linkages such that the oligonucleotides consisted ofeither full phosphorothioate backbones or mixed phosphorothioate andphosphodiester backbones (mixed backbone compounds). Modified antisensecompounds also contained sugar moiety substitutions at the 2′ position,comprising a 2′-methoxyethyl (2′-MOE) or a 2′-0-dimethylaminoethoxyethyl(2′-DMAEOE). Further modifications included nucleobase substitutions,wherein the unmodified cytosine nucleobase was used in place of themodified 5-methylcytosine at one position in the antisense compound. Thecompounds are shown in Table 4.

ISIS 145733 (SEQ ID NO: 106), ISIS 145742 (SEQ ID NO: 265) and ISIS145746 (SEQ ID NO: 266) are chimeric oligonucleotides having 2′-MOEwings and a deoxy gap with phosphorothioate linkages throughout theoligonucleotide. ISIS 257016 (SEQ ID NO: 106), ISIS 341699 (SEQ ID NO:265) and ISIS 351642 (SEQ ID NO: 266) are chimeric oligonucleotideshaving 2′-MOE wings and a deoxy gap, with phosphodiester linkages in thewings and phosphorothioate linkages in the gap. ISIS 351641 (SEQ ID NO:106), ISIS 360886 (SEQ ID NO: 106) and ISIS 360887 (SEQ ID NO: 106) arechimeric oligonucleotides having 2′-MOE wings and a deoxy gap, withphosphorothioate linkages in the gap and phosphodiester linkages in thewings, except for one phosphorothioate linkage in the wing(s) at eitherthe extreme 5′ end (ISIS 360886), the extreme 3′ end (ISIS 360887) orboth of the extreme 5′ and 3′ ends (ISIS 351641).

ISIS 323294 (SEQ ID NO: 106) consists of 2′-MOE nucleotides at positions1, 2, 3, 4, 17 and 19, 2′-DMAEOE nucleotides at positions 5, 16, 18 and20 and 2′-deoxynucleotides at positions 6 through 15, withphosphorothioate linkages throughout the oligonucleotide. ISIS 323295(SEQ ID NO: 106) consists of 2′-MOE nucleotides at positions 1, 2, 3, 4,17 and 19, 2′-DMAEOE nucleotides at positions 5, 16, 18 and 20 and2′-deoxynucleotides at positions 6 through 15, wherein the first andlast 4 internucleoside linkages are phosphodiester and the centralinternucleoside linkages are phosphorothioate.

The nucleotides in the 3′ most positions in ISIS 251017 and 257018 arecytosine residues (indicated by an asterisk in Table 4). All othercytosine residues of the oligonucleotides listed above are5-methylcytosines. The compounds are shown in Table 4. Phosphodiester(P═O) internucleoside linkages are indicated by an “o” betweennucleotide positions. Phosphorothioate (P═S) internucleoside linkagesare indicated by an “s” between nucleotide positions. 2′-MOE nucleotidesare underscored and 2′-DMAEOE nucleotides are emboldened. All compoundsin Table 4 target the coding region of murine SGLT2 (provided herein asSEQ ID NO: 11). TABLE 4 Chemical modifications of antisense compoundstargeting SGLT2 SEQ ID ISIS # Sequence NO 145733GsAsAsGsTsAsGsCsCsAsCsCsAsAsCsTsGsTsGsC 106 257016GoAoAoGoTsAsGsCsCsAsCsCsAsAsCsToGoToGoC 106 257017GsAsAsGsTsAsGsCsCsAsCsCsAsAsCsTsGsTsGsC* 106 257018GoAoAoGoTsAsGsCsCsAsCsCsAsAsCsToGoToGoC* 106 145742GsAsGsAsAsCsAsTsAsTsCsCsAsCsCsGsAsGsAsT 265 341699GoAoGoAoAsCsAsTsAsTsCsCsAsCsCsGoAoGoAoT 265 145746CsTsGsCsAsCsAsGsTsGsTsCsTsGsTsGsTsAsCsA 266 351642CoToGoCoAsCsAsGsTsGsTsCsTsGsTsGoToAoCoA 266 351641GsAoAoGoTsAsGsCsCsAsCsCsAsAsCsToGoToGsC 106 360886GsAoAoGoTsAsGsCsCsAsCsCsAsAsCsToGoToGoC 106 360887GoAoAoGoTsAsGsCsCsAsCsCsAsAsCsToGoToGsC 106 323294GsAsAsGsTsAsGsCsCsAsCsCsAsAsCsTsGsTsGsC 106 323295GoAoAoGoTsAsGsCsCsAsCsCsAsAsCsToGoToGoC 106

Example 19 Effects of Antisense Inhibition of SGLT2 in Mice: Comparisonof Various Chemistries

In accordance with the present invention, SGLT2 antisense compoundsdescribed in Example 18 were investigated for their activity in vivo.ISIS 29837 (TCGATCTCCTTTTATGCCCG, SEQ ID NO: 256) served as a controlcompound and is a chimeric oligonucleotide (“gapmer”) 20 nucleotides inlength, composed of a central “gap” region consisting of ten2′-deoxynucleotides, which is flanked on both sides (5′ and 3′directions) by five-nucleotide “wings”. The wings are composed of2′-methoxyethyl (2′-MOE) nucleotides. The internucleoside (backbone)linkages are phosphorothioate (P═S) throughout the oligonucleotide. Allcytidine residues are 5-methylcytidines.

Male 6-week old Balb/c mice (Charles River Laboratories, Wilmington,Mass.) were given intraperitoneal injections of ISIS 145733, ISIS257016, ISIS 323294, ISIS 323295 or ISIS 29837 at a dose of 25 mg/kgtwice per week for two weeks. Saline-injected animals also served as acontrol. Each treatment group contained four animals. The mice weresacrificed 2 days following administration of the fourth and final doseof oligonucleotide or saline.

Mice were evaluated for SGLT2 levels in kidney. Target levels weredetermined by quantitative real-time PCR as described by other examplesherein. PCR results were normalized to the ubiquitously expressed mousecyclophilin A gene.

Probes and primers to mouse SGLT2 were designed to hybridize to a mouseSGLT2 sequence, using published sequence information (incorporatedherein as SEQ ID NO: 11). For mouse SGLT2 the PCR primers were: (SEQ IDNO: 257) forward primer: CTCGTCTCATACCCGAGTTCTTCT (SEQ ID NO: 258)reverse primer: AATGATGGCGAAATAGAGGTAGTGTAC and the PCR probe was: (SEQID NO: 259) FAM-TGCGACCCTCAGCGTGCCC-TAMRA

where FAM is the fluorescent dye and TAMRA is the quencher dye. Formouse cyclophilin A the PCR primers were: (SEQ ID NO: 260) forwardprimer: TCGCCGCTTGCTGCA (SEQ ID NO: 261) reverse primer:ATCGGCCGTGATGTCGA and the PCR probe was: (SEQ ID NO: 262)5′ JOE-CCATGGTCAACCCCACCGTGTTC-3′where JOE is the fluorescent reporter dye and TAMRA is the quencher dye.

The data are expressed as percent change (“−” indicates a decrease)relative to saline treated animals and are shown in Table 5. TABLE 5Antisense inhibition of SGLT2 mRNA expression in vivo by 25 mg/kg dosesof antisense compounds % change in SGLT2 expression relative to salineISIS ISIS ISIS ISIS ISIS 145733 257016 323294 323295 29837 −44 −82 −40−31 −23

These data illustrate that antisense compounds of different chemistriesinhibit the expression of SGLT2 mRNA in mouse kidney.

Mice were further evaluated for total body weight, liver weight andspleen weight. Significant changes in spleen, liver or body weight canindicate that a particular compound causes toxic effects. The data areexpressed as percent change (“+” indicates an increase, “−” indicates adecrease) relative to saline control. The results are presented in Table6. TABLE 6 Effects of antisense compounds on total body weight, liverweight and spleen weight in mice Weight as % change relative to salinecontrol 145733 257016 323294 323295 29837 Total Body 0 0 −1 −3 0 Liver+1 +1 +9 +4 +12 Spleen +4 +1 +19 +8 +1

All changes were within the margin of error of the experiment. Nosignificant changes in body weight were observed during the treatment orat study termination. No significant changes in liver or spleen weightswere observed.

Toxic effects of compounds administered in vivo can also be assessed bymeasuring the levels of enzymes and proteins associated with disease orinjury of the liver or kidney. Elevations in the levels of the serumtransaminases aspartate aminotransferase (AST) and alanineaminotransferase (ALT) are often indicators of liver disease or injury.Serum total bilirubin is an indicator of liver and biliary function, andalbumin and blood urea nitrogen (BUN) are indicators of renal function.Glucose and triglyceride levels are sometimes altered due to toxicity ofa treatment. Serum glucose also depends in part upon the activity ofSGLT2.

In accordance with the present invention, the levels of ALT, AST, totalbilirubin, albumin, BUN, glucose and triglyceride were measured in micetreated with the compounds of the invention. Serum was analyzed byLabCorp Testing Facility (San Diego, Calif.). The results are expressedas units measured and are shown in Table 7. TABLE 7 Effects of antisensecompounds targeting SGLT2 on liver and kidney function in mice SerumNormal Treatment and units measured indicator Range Saline 145733 257016323294 323295 29837 BUN mg/dL 15-40 27 29 33 29 30 30 Albumin 2.5-4.0 33 3 3 3 3 g/dL Bilirubin mg/dL 0.1-1.0 0.1 0.1 0.1 0.1 0.1 0.1 AST 30-300 124 83 129 174 89 114 IU/L ALT  30-200 33 26 47 61 32 31 IU/LTriglycerides  25-100* 179 154 157 160 209 198 mg/dL Glucose  80-150*242 270 222 284 271 235 mg/dL*Triglyceride and glucose levels are routinely higher in the Balb/cstrain of mice than in other strains of mice.

The levels of routine clinical indicators of liver and kidney injury anddisease are within normal ranges and are not significantly changedrelative to saline-treated animals, demonstrating that the compounds ofthe invention do not significantly affect renal or hepatic function.Triglyceride and glucose levels, while outside the normal range for mostmice as is common in the Balb/c strain, are not significantly elevatedrelative to saline-treated animals.

Mice injected with ISIS 145733, 257016, 323294 and 323295 were alsoevaluated histologically following routine procedures. Liver, spleen,kidney, intestine, pancreas, lung, skin, heart and muscle samples wereprocured, fixed in 10% neutral-buffered formalin and processed forstaining with hematoxylin and eosin, to visualize nuclei and cytoplasm,or with the anti-oligonucleotide IgG1 antibody 2E1-B5 (Berkeley AntibodyCompany, Berkeley, Calif.) to assess oligonucleotide staining patterns.Hematoxylin and eosin staining in most tissues exhibited no significantdifference between saline- and oligonucleotide-treated animals. Heartsections from animals treated with 323294 and 323295 showed a highamount of inflammation relative to hearts from saline-treated mice.2E1-B5 antibody was recognized using an isospecific anti-IgG2horse-radish peroxidase-conjugated secondary antibody (Zymed, SanFrancisco, Calif.) and immunostaining was developed with3,3′-diaminobenzidene (DAKO, Carpenteria, Calif.). 2E1-B5 staining wasperformed in duplicate and showed that none of the chemistriessignificantly stained the liver, while staining was observed in thekidney proximal tubules.

The results illustrated in this example demonstrate that antisensecompounds of different chemistries are delivered to the kidney, reduceSGLT2 expression in vivo, and that treatment with these compounds doesnot result in liver or kidney toxicity.

Example 20 Effects of Antisense Compounds on SGLT2 mRNA Expression invivo: Dose Response Study Comparing Mixed Backbone and FullPhosphorothioate Backbones

ISIS 145733 and ISIS 257016 were selected for a dose response study inmice. Male 8-week old Balb/c mice (Charles River Laboratories,Wilmington, Mass.) were given intraperitoneal injections of either ISIS145733 or ISIS 257016 at doses of 6.25, 12.5, 25 and 50 mg/kg twice perweek for two weeks. Saline-injected animals served as controls. A totalof 4 animals were injected per group. The mice were sacrificed 2 daysfollowing administration of the fourth and final dose of oligonucleotideor saline.

Mice were evaluated for SGLT2 levels in kidney. Target levels weredetermined by quantitative real-time PCR as described by other examplesherein. PCR results were normalized to cyclophilin as described inExample 19. The data are expressed as percent change (“+” indicates anincrease, “−” indicates a decrease) relative to saline treated animalsand are illustrated in Table 8. TABLE 8 Antisense inhibition of SGLT2mRNA expression in vivo by antisense compounds with varying chemistries% change in SGLT2 expression relative to saline Dose of oligonucleotideISIS ISIS mg/kg 145733 257016 6.25 −3 −58 12.5 −7 −68 25 −37 −68 50 −34−77

These results illustrate that the compounds of the invention, both fullphosphorothioate and mixed backbone oligonucleotides, inhibit theexpression of SGLT2 in vivo in a dose-dependent manner.

The levels of SGLT2 expression were also evaluated by Northern blotanalysis of both pooled and individual RNA samples, to validate thetarget reduction observed by real-time PCR. Total RNA was prepared fromprocured tissues of sacrificed mice by homogenization in GITC buffer(Invitrogen, Carlsbad, Calif.) containing 2-mercaptoethanol(Sigma-Aldrich, St. Louis, Mo.) following manufacturer's recommendedprotocols followed by ultracentrifugation through a CsCl cushion. Twentymicrograms of total RNA was fractionated by electrophoresis through 1.2%agarose gels containing 1.1% formaldehyde using a MOPS buffer system(AMRESCO, Inc. Solon, Ohio). RNA was transferred from the gel toHYBOND™-N+ nylon membranes (Amersham Pharmacia Biotech, Piscataway,N.J.) by overnight capillary transfer. RNA transfer was confirmed by UVvisualization. Membranes were fixed by UV cross-linking using aSTRATALINKER™ UV Crosslinker 2400 (Stratagene, Inc, La Jolla, Calif.)and then probed using RapidHYB™ hybridization solution (AmershamPharmacia Biotech, Piscataway, N.J.) using manufacturer'srecommendations for stringent conditions.

To detect mouse SGLT2, a mouse SGLT2 specific template was prepared byPCR using the forward primer 5′-ATGGAGCAACACGTAGAGGCAGGCT-3′ (SEQ ID NO:263) and the reverse primer 5′-GAGTGCCGCCAGCCCTCCTGTCACA-3′ (SEQ ID NO:264) and gel purified. The probe was prepared by asymmetric PCR with thepurified template and the reverse primer incorporating ³²p CTP to labelthe probe. Following hybridization blots were exposed overnight tophosphorimager screens (Molecular Dynamics, Amersham) and quantitated.To normalize for variations in loading and transfer efficiency membraneswere stripped and probed for mouse glyceraldehyde-3-phosphatedehydrogenase (GAPDH) RNA (Clontech, Palo Alto, Calif.).

For pooled sample analysis, equal amounts of RNA isolated from thekidneys of mice in the same treatment was combined for a total of 20 μg,and the pooled sample was subjected to Northern blot analysis. Theresults of the pooled sample analysis are shown in Table 9 and arenormalized to saline controls (“+” indicates an increase, “−” indicatesa decrease). TABLE 9 Northern Analysis of SGLT2 message in pooled kidneyRNA samples % change in SGLT2 expression Dose of relative to salineoligonucleotide ISIS ISIS mg/kg 145733 257016 6.25 +21 −57 12.5 +7 −5025 −35 −75 50 −35 −82

These results demonstrate that, as determined by Northern blot analysisof pooled samples, ISIS 257016 inhibits SGLT2 expression inhibits SGLT2expression at all doses of antisense compound in a dose-dependentmanner, where as ISIS 145733 inhibits SLGT2 expression at the twohighest doses of antisense compound.

Target levels in kidney RNA samples from individual mice were alsomeasured by Northern blot analysis. Equal amounts of RNA wereindividually subjected to Northern blot analysis to determine the levelof SGLT2. Target level measurements for each treatment group were thenaveraged. The results are shown in Table 10 and are normalized to salinecontrols (“−” indicates a decrease). TABLE 10 Northern analysis of SGLT2message in individually measured RNA samples % change in SGLT2expression Dose of relative to saline oligonucleotide ISIS ISIS mg/kg145733 257016 6.25 −34 −66 12.5 −38 −68 25 −39 −74 50 −59 −82

Treated mice were further evaluated at the end of the treatment periodfor total body, liver and spleen weight. The data are expressed aspercent change (“+” indicates an increase, “−” indicates a decrease)relative to saline control. The results are presented in Table 11 TABLE11 Effects of antisense compounds on total body weight, liver weight andspleen weight in mice % Change relative to saline-treated ISIS ISIS Doseof 145733 257016 oligonucleotide Total Total mg/kg Body Liver SpleenBody Liver Spleen 6.25 −4 −10 −12 −1 −3 +1 12.5 −6 −2 −7 −3 −13 −9 25 1−1 +10 1 −8 +8 50 −1 +6 +10 −3 −9 +12

These data demonstrate that no significant changes in total body, liveror spleen weights are observed following treatment with ISIS 145733 orISIS 257016 at 4 different doses. No changes in total body weight wereobserved during the treatment period, or at study termination.

In addition to the indicators of toxicity listed in Example 19,creatinine levels are also used to evaluate renal function. Inaccordance with the present invention, the levels of ALT, AST, totalbilirubin, creatinine, BUN, glucose and triglyceride were measured inmice treated with the compounds of the invention. Serum was analyzed byLabCorp Testing Facility (San Diego, Calif.). The results are expressedas units measured and are shown in Table 12. TABLE 12 Effects ofantisense compounds targeting SGLT2 on liver and kidney function in miceUnits measured per treatment and dose Serum Normal 145733 indicatorRange Saline 25 mg/kg 145733 50 mg/kg 257016 25 mg/kg 257016 50 mg/kgBUN 15-40 24 24 25 26 26 mg/dL Creatinine 0.0-1.0 0.1 0.1 0.1 0.125 0.1mg/L Bilirubin mg/dL 0.1-1.0 0.125 0.1 0.1 0.1 0.1 AST  30-300 77 65 96133 141 IU/L ALT  30-200 24 18 22 34 35 IU/L Triglycerides  25-100* 165169 230 130 111 mg/dL Glucose  80-150* 236 280 256 244 248 mg/dL*Triglyceride and glucose levels are routinely higher in the Balb/cstrain of mice than in other strains of mice.

The AST levels in animals treated with 25 mg/kg of ISIS 145733 areslightly below the normal range, as is the ALT level for saline treatedmice. Otherwise, the levels of routine clinical indicators of liver andkidney injury and disease are within normal ranges and are notsignificantly changed relative to saline-treated animals, demonstratingthat the compounds of the invention do not significantly affect renal orhepatic function. Triglyceride and glucose levels, while outside thenormal range as is common in the Balb/c strain, are not significantlyelevated relative to saline-treated animals.

Mice injected with ISIS 145733 and 257016 at doses from 6.25 to 50 mg/kgwere also evaluated histologically following routine procedures. Liverand kidney samples were procured, fixed in 10% neutral-buffered formalinand processed for staining with hematoxylin and eosin, to visualizenuclei and cytoplasm, or with the anti-oligonucleotide IgG1 antibody2E1-B5 (Berkeley Antibody Company, Berkeley, Calif.) to assessoligonucleotide staining patterns. Hematoxylin and eosin stainingexhibited no significant difference between saline- andoligonucleotide-treated animals. 2E1-B5 antibody was recognized using anisospecific anti-IgG2 horseradish peroxidase-conjugated secondaryantibody (Zymed, San Francisco, Calif.) and immunostaining was developedwith 3,3′-diaminobenzidene (DAKO, Carpenteria, Calif.). 2E1 stainingshowed no detectable oligonucleotide in the liver, while staining wasobserved in the kidney proximal tubules. Staining intensity lessenedconcomitantly with a decrease in oligonucleotide dose.

The results illustrated in this example demonstrate that antisensecompounds of different chemistries are delivered to the kidney, reduceSGLT2 expression in vivo in a dose-dependent manner, and that treatmentwith these compounds does not result in liver or kidney toxicity.

Example 21 Effects of Antisense Compounds on SGLT2 mRNA Expression invivo: an Additional Dose Response Study Comparing Mixed Backbone andFull Phosphorothioate Backbones

ISIS 145733 and ISIS 257016 were selected for a dose response study inmice using two identical and two lower doses with respect to the dosesused in Example 20.

Male 8-week old Balb/c mice (Charles River Laboratories, Wilmington,Mass.) were given intraperitoneal injections of ISIS 145733 or ISIS257016 at doses of 1, 5, 25 or 50 mg/kg twice per week for two weeks.Saline-injected animals served as a control. In addition, as aspecificity control, the same doses of SGLT2 antisense oligomericcompounds do not significantly inhibit expression of SGLT1 mRNA inkidney cells. Each treatment group contained 4 mice. The mice weresacrificed 2 days following administration of the fourth and final doseof oligonucleotide or saline.

Mice were evaluated for SGLT2 levels in kidney and liver. Target levelswere determined by quantitative real-time PCR as described by otherexamples herein. PCR results were normalized to cyclophilin. The dataare expressed as percent change relative to saline treated animals (“+”indicates an increase, “−” indicates a decrease) and are illustrated inTable 13. TABLE 13 Antisense inhibition of SGLT2 mRNA expression in vivoby antisense compounds with varying chemistries % change in SGLT2expression relative to saline Dose of Kidney Liver oligonucleotide ISISISIS ISIS ISIS mg/kg 145733 257016 145733 257016 1 +2 −46 −19 +13 5 −15−64 −39 +1 25 −34 −74 −21 −5 50 −40 −76 −59 −12

These results illustrate that the compounds of the invention, both fullphosphorothioate and mixed backbone oligonucleotides, can inhibit theexpression of kidney SGLT2 in a dose-dependent manner. Greaterinhibition is observed in kidneys from mice treated with ISIS 257016, amixed backbone antisense compound. SGLT2 is not highly expressed inliver, therefore target levels are low before treatment and thereforemore difficult to accurately measure. While ISIS 145733 and ISIS 257016also lowered liver SGLT2 expression, with 145733 having a greater effectin liver than the mixed backbone ISIS 257016.

Treated mice were further evaluated for liver and spleen weight. Thedata are expressed as percent change (“+” indicates an increase, “−”indicates a decrease) relative to saline control. The results arepresented in Table 14. TABLE 14 Effects of antisense compounds on totalbody weight, liver weight and spleen weight in mice % change in body,liver and spleen weight ISIS ISIS Dose of 145733 257016 oligonucleotideTotal Total mg/kg Body Liver Spleen Body Liver Spleen 1 0 −6 +10 −2 −8+13 5 +3 +1 +10 −3 −9 +5 25 −1 +2 −4 +2 +2 +12 50 −1 +13 +35 −2 −6 +15

No significant change was observed in total body weight at timepointsthroughout or at the termination of the study. Treatments of 25 mg/kgISIS 145733 and 50 mg/kg 257016 resulted in a decrease and increase inliver weight, respectively, however, these changes are within the marginof error for the data and are therefore not significant.

In addition to the other serum markers described herein, cholesterollevels can be used as a measure of toxicity. In accordance with thepresent invention, the levels of ALT, AST, total bilirubin, albumin,creatinine, BUN, triglyceride, cholesterol and glucose were measured inmice treated with the compounds of the invention. Plasma samples wereanalyzed using the Olympus AU400e automated chemistry analyzer (OlympusAmerica, Irving, Tex.). The results are expressed as units measured areshown for ISIS 145733 in Table 15 and for ISIS 257016 in Table 16. TABLE15 Effects of the full phosphorothioate antisense compound ISIS 145733on indicators of liver and kidney function Units measured per Normaldose of ISIS 145733 Serum indicator Range Saline 1 mg/kg 5 mg/kg 25mg/kg 50 mg/kg BUN 15-40 27 31 31 30 25 mg/dL Creatinine 0.0-1.0 0.2 0.20.2 0.2 0.2 mg/L Bilirubin mg/dL 0.1-1.0 0.3 0.2 0.1 0.3 0.1 AST  30-30092 91 45 133 56 IU/L ALT  30-200 35 27 26 37 31 IU/L Albumin 2.5-4.0 3 33 3 3 g/dL Triglycerides mg/dL  25-100* 136 188 183 153 224 Cholesterol 70-125 122 116 117 120 132 mg/dL Glucose  80-150* 208 202 173 170 161mg/dL

TABLE 16 Effects of the mixed backbone antisense compound ISIS 257016 onindicators of liver and kidney function Units measured per Normal doseof ISIS 257016 Serum indicator Range Saline 1 mg/kg 5 mg/kg 25 mg/kg 50mg/kg BUN 15-40 27 23 29 25 28 mg/dL Creatinine 0.0-1.0 0.2 0.2 0.2 0.20.2 mg/L Bilirubin mg/dL 0.1-1.0 0.3 0.2 0.2 0.2 0.2 AST  30-300 92 7473 99 138 IU/L ALT  30-200 35 34 34 46 48 IU/L Albumin 2.5-4.0 3 3 3 3 3g/dL Triglycerides mg/dL  25-100* 136 271 233 225 136 Cholesterol 70-125 122 116 124 144 137 mg/dL Glucose  80-150* 208 180 178 154 182mg/dL*Triglyceride and glucose levels are routinely higher in the Balb/cstrain of mice than in other strains of mice.

The levels of routine clinical indicators of liver and kidney injury anddisease are within normal ranges and are not significantly changedrelative to saline-treated animals, demonstrating that the compounds ofthe invention do not significantly affect renal or hepatic function.Triglyceride and glucose levels, while outside the normal range as iscommon in the Balb/c strain, are not significantly elevated relative tosaline-treated animals.

Mice injected ISIS 145733 and 257016 at 1-50 mg/kg were also evaluatedhistologically following routine procedures. Liver and kidney sampleswere procured, fixed in 10% neutral-buffered formalin and processed forstaining with hematoxylin and eosin, to visualize nuclei and cytoplasm,or with the anti-oligonucleotide IgG1 antibody 2E1-B5 (Berkeley AntibodyCompany, Berkeley, Calif.) to assess oligonucleotide staining patterns.Hematoxylin and eosin staining in most tissues exhibited no significantdifference between saline- and 145733-treated animals, with theexception of slight inflammatory cell infiltration in the liver tissue.Livers from mice treated with ISIS 257016 showed evidence of nucleardegradation and mitosis at 50 mg/kg and slight mitosis at 25 mg/kg.Kidneys from ISIS 257016 exhibited no significant differences comparedto saline-treated kidneys. 2E1-B5 antibody was recognized using anisospecific anti-IgG2 horse-radish peroxidase-conjugated secondaryantibody (Zymed, San Francisco, Calif.) and immunostaining was developedwith 3,3′-diaminobenzidene (DAKO, Carpenteria, Calif.). Staining withthe 2E1 antibody showed weak staining in liver and kidneys from animalstreated with ISIS 145733, whereas staining was strong in liver andkidney from animals treated with ISIS 257016. Kidney 2E1 stainingappears in a punctate pattern.

Example 22 Dose Response Study Comparing Mixed Backbone and FullPhosphorothioate Backbones: a Second SGLT2 Antisense Sequence

A second mixed backbone SGLT2 oligonucleotide, ISIS 341699 (SEQ ID NO:265), and control phosphorothioate SGLT2 oligonucleotide, ISIS 145742(SEQ ID NO: 265), were selected for a dose response study in mice. Forcomparison, ISIS 257016 (mixed backbone; SEQ ID NO: 106) also wasincluded in this study.

Male 8-week old Balb/c mice (Charles River Laboratories, Wilmington,Mass.) were given intraperitoneal injections of ISIS 341699, ISIS 145742or ISIS 257016 twice per week for two weeks with the doses shown inTable 17. Saline-injected animals served as controls. Each treatmentgroup contained 4 mice. The mice were sacrificed 2 days followingadministration of the fourth and final dose of oligonucleotide orsaline.

Mice were evaluated for SGLT2 levels in kidney. Target levels weredetermined by quantitative real-time PCR as described by other examplesherein. PCR results were normalized to cyclophilin. The data areexpressed as percent change relative to saline treated animals (“+”indicates an increase, “−” indicates a decrease) and are illustrated inTable 17. TABLE 17 Antisense inhibition of SGLT2 mRNA expression in vivoby mixed backbone and full phosphorothioate oligonucleotides (expressedas percent change in SGLT2 mRNA expression relative to saline) Dose ofoligonucleotide ISIS ISIS ISIS mg/kg 145742 341699 257016 0.2 — — −18.91 — −1.8 −50.5 5 −0.6 −10.9 −56.7 25 −24.9 −23.9 — 50 −32.6 — —

These results illustrate that the compounds of the invention, both fullphosphorothioate and mixed backbone oligonucleotides, can inhibit theexpression of kidney SGLT2 in a dose-dependent manner. However, lowerdoses of the mixed backbone compound are required to inhibit SGLT2expression in kidneys from treated mice.

Treated mice were further evaluated for liver and spleen weight. Thedata are expressed as percent change in body or organ weight (“+”indicates an increase, “−” indicates a decrease). The results arepresented in Table 18 and Table 19. TABLE 18 Effects of antisensecompounds on total body weight of mice (expressed as percent change inbody weight) Dose of oligonucleotide ISIS ISIS ISIS mg/kg 145742 341699257016 0.2 — — +7.9 1 — +5.7 +5.8 5 +5.0 +5.8 +3.2 25 +2.0 +2.5 — 50+7.2 — —

TABLE 19 Effects of antisense compounds on liver weight and spleenweight of mice (expressed as percent change in organ weight) Dose ofLiver Spleen oligonucleotide ISIS ISIS ISIS ISIS ISIS ISIS mg/kg 145742341699 257016 145742 341699 257016 0.2 — — −6.0 — — −4.7 1 — +2.3 +14.9— −4.2 +1.4 5 +7.1 +2.2 +7.0 +10.6 −2.8 −7.6 25 +7.2 +5.8 — +0.8 −0.2 —50 +12.1 — — +9.4 — —

No significant change was observed in total body weight, liver weight orspleen weight at timepoints throughout or at the termination of thestudy.

Levels of BUN, creatinine, AST, ALT, albumin, triglycerides, cholesteroland glucose were measured in mice treated with the compounds of theinvention. Plasma samples were analyzed using the Olympus AU400eautomated chemistry analyzer (Olympus America, Irving, Tex.). Theresults, expressed as units measured, are shown for ISIS 145742 in Table20, ISIS 341699 in Table 21 and ISIS 257016 in Table 22. TABLE 20 Effectof the full phosphorothioate antisense compound ISIS 145742 onindicators of liver and kidney function Units measured per Serum Normaldose of ISIS 145742 indicator Range Saline 5 mg/kg 25 mg/kg 50 mg/kg BUN15-40 20 21.3 25.5 20.8 mg/dL Creatinine 0.0-1.0 0.1 0.2 0.2 0.2 mg/LAST  30-300 113 75.3 83.5 145.3 IU/L ALT  30-200 35.5 29.8 40.3 47.5IU/L Albumin 2.5-4.0 3.0 3.0 2.9 2.9 g/dL Triglycerides  25-100* 223.8176.5 192 176.8 mg/dL Cholesterol  70-125 129 119.5 119.5 113.5 mg/dLGlucose  80-150* 176.5 196.5 192 194.8 mg/dL

TABLE 21 Effect of mixed backbone antisense compound ISIS 341699 onindicators of liver and kidney function Units measured per Normal doseof ISIS 341699 Serum indicator Range Saline 1 mg/kg 5 mg/kg 25 mg/kg BUN15-40 20 20 21.8 22 mg/dL Creatinine 0.0-1.0 0.1 0.2 0.2 0.2 mg/L AST 30-300 113 78.2 119 64.8 IU/L ALT  30-200 35.5 36.2 37.3 33.0 IU/LAlbumin 2.5-4.0 3.0 3.3 3.1 3.2 g/dL Triglycerides  25-100* 223.8 206.4186.8 183.5 mg/dL Cholesterol  70-125 129 135 124 120.8 mg/dL Glucose 80-150* 176.5 203.2 171.5 197 mg/dL

TABLE 22 Effect of mixed backbone antisense compound ISIS 257016 onindicators of liver and kidney function Units measured per Normal doseof ISIS 257016 Serum indicator Range Saline 0.2 mg/kg 1 mg/kg 5 mg/kgBUN 15-40 20 21.8 26.3 20.5 mg/dL Creatinine 0.0-1.0 0.1 0.2 0.2 0.2mg/L AST  30-300 113 123.8 85.3 69.5 IU/L ALT  30-200 35.5 36.8 44 43IU/L Albumin 2.5-4.0 3.0 3.1 3.4 3.1 g/dL Triglycerides  25-100* 223.8138.8 268.3 212.8 mg/dL Cholesterol  70-125 129 128 152 135.3 mg/dLGlucose  80-150* 176.5 208.8 212.3 164.5 mg/dL*Triglyceride and glucose levels are routinely higher in the Balb/cstrain of mice than in other strains of mice.

In some oligonucleotide-treated animals cholesterol levels were abovethe normal range; however, this elevation is not significant sincesaline-treated animals also exhibited cholesterol above the normalrange. The levels of the remaining routine clinical indicators of liverand kidney injury and disease are within normal ranges and are notsignificantly changed relative to saline-treated animals, demonstratingthat the compounds of the invention do not significantly affect renal orhepatic function. Triglyceride and glucose levels, while outside thenormal range as is common in the Balb/c strain, are not significantlyelevated relative to saline-treated animals.

Mice injected with ISIS 145742, ISIS 341699 and ISIS 257016 at 0.2-50mg/kg were also evaluated histologically following routine procedures.Liver and kidney samples were procured, fixed in 10% neutral-bufferedformalin and processed for staining with hematoxylin and eosin or withthe anti-oligonucleotide IgGI antibody 2E1-B5, as described in otherexamples herein. Hematoxylin and eosin staining in both liver and kidneytissues exhibited no significant difference between saline- andantisense oligonucleotide-treated animals. Staining with the 2E1antibody showed high background in sinusoidal tissues of liver from thesaline-injected animals, therefore making it difficult to interpretpositive staining in the oligonucleotide-treated livers. Kidney samplesfrom saline-injected animals and animals treated with 0.2 mg/kg ISIS257016 showed no positive oligonucleotide staining; however, theremainder of the oligonucleotide-treated animals demonstrated highlevels of staining in the proximal tubules, which increased with dose.

The results illustrated in this example demonstrate that antisensecompounds of different chemistries are delivered to the kidney, reduceSGLT2 expression in vivo in a dose-dependent manner, and that treatmentwith these compounds does not result in liver or kidney toxicity. Theresults further demonstrate that mixed backbone compounds ISIS 341699and ISIS 257016 are particularly effective at reducing target mRNAlevels in the kidney.

Example 23 Dose Response Study Comparing Mixed Backbone and FullPhosphorothioate Backbones: a Third SGLT2 Antisense Sequence

A third mixed backbone SGLT2 oligonucleotide, ISIS 351642 (SEQ ID NO:266), and control phosphorothioate SGLT2 oligonucleotide, ISIS 145746(SEQ ID NO: 266), were selected for a dose response study in mice.

Male 7-week old Balb/c mice (Charles River Laboratories, Wilmington,Mass.) were given intraperitoneal injections of ISIS 145746 or ISIS351642 twice per week for two weeks with the doses shown in Table 23.Saline-injected animals served as controls. Each treatment groupcontained 4 mice. The mice were sacrificed 2 days followingadministration of the fourth and final dose of oligonucleotide orsaline.

Mice were evaluated for SGLT2 levels in kidney. Target levels weredetermined by quantitative real-time PCR as described by other examplesherein. PCR results were normalized to cyclophilin. The data areexpressed as percent change relative to saline treated animals (“+”indicates an increase, “−” indicates a decrease) and are illustrated inTable 23. TABLE 23 Antisense inhibition of SGLT2 mRNA expression in vivoby mixed backbone and full phosphorothioate oligonucleotides (expressedas percent change in SGLT2 mRNA expression relative to saline) Dose ofoligonucleotide ISIS ISIS mg/kg 145746 351642 1 — −26.7 5 −5.8 −35.1 25−10.5 −44.3 50 −35.6 −31.8

These results illustrate that the compounds of the invention, both fullphosphorothioate and mixed backbone oligonucleotides, can inhibit theexpression of kidney SGLT2 in a dose-dependent manner. At doses of 5 and25 mg/kg, greater inhibition is observed in kidneys from mice treatedwith ISIS 351462, suggesting the mixed backbone antisense compound is amore efficient inhibitor of target mRNA expression in the kidney.

Treated mice were further evaluated for body weight, liver weight andspleen weight. The data are expressed as percent change in body or organweight (“+” indicates an increase, “−” indicates a decrease). Theresults are presented in Table 24. TABLE 24 Effects of antisensecompounds on total body weight, liver weight and spleen weight of micePercent change in weight ISIS ISIS Dose of 145746 351642 oligonucleotideTotal Total mg/kg Body Liver Spleen Body Liver Spleen 1 — — — +6.9 −8.2+0.8 5 +3.6 −5.7 +6.5 +4.6 −0.6 −7.9 25 +5.4 −2.0 +3.7 +4.7 −10.6 +1.150 +12.1 −8.4 +10.0 +7.4 −3.0 +1.3

No significant change was observed in total body weight, liver weight orspleen weight at timepoints throughout or at the termination of thestudy.

Levels of BUN, creatinine, AST, ALT, albumin, triglycerides, cholesteroland glucose were measured in mice treated with the compounds of theinvention. Plasma samples were analyzed using the Olympus AU400eautomated chemistry analyzer (Olympus America, Irving, Tex.). Theresults, expressed as units measured, are shown for ISIS 145746 in Table25 and ISIS 351642 in Table 26. TABLE 25 Effect of the fullphosphorothioate antisense compound ISIS 145746 on indicators of liverand kidney function Units measured per dose of ISIS 145746 Serum NormalSa- 1 5 25 50 indicator Range line mg/kg mg/kg mg/kg mg/kg Creatinine0.0-1.0 0.1 — 0.2 0.2 0.1 mg/L AST  30-300 129 — 60 84 155 IU/L ALT 30-200 30 — 28 26 77 IU/L Albumin 2.5-4.0 2.8 — 2.9 2.8 2.9 g/dLTriglycerides  25-100* 298 — 268 259 236 mg/dL Cholesterol  70-125 116 —118 108 106 mg/dL Glucose  80-150* 163 — 162 181 179 mg/dL

TABLE 26 Effect of mixed backbone antisense compound ISIS 351642 onindicators of liver and kidney function Units measured per dose of ISIS351642 Serum Normal Sa- 1 5 25 50 indicator Range line mg/kg mg/kg mg/kgmg/kg Creatinine 0.0-1.0 0.1 0.1 0.1 0.2 0.2 mg/L AST  30-300 129 132 75131 160 IU/L ALT  30-200 30 31 28 29 31 IU/L Albumin 2.5-4.0 2.8 2.9 3.02.7 2.8 g/dL Triglycerides  25-100* 298 238 287 240 233 mg/dLCholesterol  70-125 116 117 122 106 113 mg/dL Glucose  80-150* 163 195175 164 171 mg/dL*Triglyceride and glucose levels are routinely higher in the Balb/cstrain of mice than in other strains of mice.

The levels of routine clinical indicators of liver and kidney injury anddisease are within normal ranges and are not significantly changedrelative to saline-treated animals, demonstrating that the compounds ofthe invention do not significantly affect renal or hepatic function.Triglyceride and glucose levels, while outside the normal range as iscommon in the Balb/c strain, are not significantly elevated relative tosaline-treated animals.

The results illustrated in this example demonstrate that antisensecompounds of different chemistries are delivered to the kidney, reduceSGLT2 expression in vivo in a dose-dependent manner, and that treatmentwith these compounds does not result in liver or kidney toxicity. Theresults further suggest that mixed backbone compound ISIS 351642 is moreeffective than full phosphorothioate oligonucleotides at reducing targetmRNA levels in the kidney, particularly at low doses.

Example 24 Comparison of a Standard Mixed Backbone Compound and a MixedBackbone Compound with Phosphorothioate Linkages at the Extreme 5′ and3′ Ends: a Single Dose Study

In accordance with the present invention, ISIS 257016 (SEQ ID NO: 106)and ISIS 351641 (SEQ ID NO: 106) were analyzed for their ability toinhibit SGLT2 expression in vivo. ISIS 257016 is a standard mixedbackbone compound having 2′-MOE wings and a deoxy gap, withphosphodiester linkages in the wings and phosphorothioate linkages inthe gap. ISIS 351641 differs from the standard mixed backbone compoundsby having one phosphorothioate linkage at each of the extreme 5′ and 3′ends of the wings.

Male 8-week old Balb/c mice (Charles River Laboratories, Wilmington,Mass.) were given a single intraperitoneal injection of ISIS 257016 orISIS 351641 at a dose of 1, 5, 25 or 50 mg/kg. Saline-injected animalsserved as controls. Each treatment group contained 4 mice. The mice weresacrificed 2 days following administration of the single dose ofoligonucleotide or saline.

Mice were evaluated for SGLT2 levels in kidney. Target levels weredetermined by quantitative real-time PCR as described by other examplesherein. PCR results were normalized to cyclophilin. The data areexpressed as percent change relative to saline treated animals (“+”indicates an increase, “−” indicates a decrease) and are illustrated inTable 27. TABLE 27 Antisense inhibition of SGLT2 mRNA expression in vivoby mixed backbone oligonucleotides (expressed as percent change in SGLT2mRNA expression relative to saline) Dose of oligonucleotide ISIS ISISmg/kg 257016 351641 1 −21.5 −14.0 5 −26.4 −19.3 25 −24.2 −12.5 50 −36.3−22.0

These results illustrate that mixed backbone compounds of the invention,with either complete phosphodiester linkages in the wings, or with theextreme 5′ and 3 ′ ends substituted with phosphorothioate linkages,inhibit the expression of kidney SGLT2 in a dose-dependent manner.However, greater inhibition is observed in kidneys from mice treatedwith ISIS 257016, which contains all phosphodiester linkages in thewings.

Treated mice were further evaluated for body weight and liver and spleenweight. The data are expressed as percent change in body or organ weight(“+” indicates an increase, “−” indicates a decrease). The results arepresented in Table 28. TABLE 28 Effects of antisense compounds on totalbody weight, liver weight and spleen weight of mice Percent change inweight ISIS ISIS Dose of 257016 351641 oligonucleotide Total Total mg/kgBody Liver Spleen Body Liver spleen 1 −0.9 +1.2 −1.6 +2.8 +3.0 −0.1 5−5.1 +5.4 +20.1 +4.0 +2.1 +9.7 25 −1.1 +3.5 +3.8 −0.7 +9.3 +5.9 50 −2.5−2.3 +7.8 +0.9 −0.7 +10.2

No significant change was observed in total body weight, liver weight orspleen weight at timepoints throughout or at the termination of thestudy.

Levels of creatinine, AST, ALT, albumin, triglycerides, cholesterol andglucose were measured in mice treated with the compounds of theinvention. Plasma samples were analyzed using the Olympus AU400eautomated chemistry analyzer (Olympus America, Irving, Tex.). Theresults, expressed as units measured, are shown for ISIS 257016 in Table29 and for ISIS 351641 in Table 30. TABLE 29 Effect of mixed backboneantisense compound ISIS 257016 on indicators of liver and kidneyfunction Units measured per dose of ISIS 257016 Serum Normal Sa- 1 5 2550 indicator Range line mg/kg mg/kg mg/kg mg/kg Creatinine 0.0-1.0 0.00.0 0.0 0.0 0.2 mg/L AST  30-300 141 62 77 89 88 IU/L ALT  30-200 30 2928 27 33 IU/L Albumin 2.5-4.0 2.9 2.8 2.8 3.0 2.9 g/dL Triglycerides 25-100* 213 253 255 347 245 mg/dL Cholesterol  70-125 118 111 116 125120 mg/dL Glucose  80-150* 155 186 172 174 169 mg/dL

TABLE 30 Effect of mixed backbone antisense compound ISIS 351641 onindicators of liver and kidney function Units measured per dose of ISIS351641 Serum Normal Sa- 1 5 25 50 indicator Range line mg/kg mg/kg mg/kgmg/kg Creatinine 0.0-1.0 0.0 0.2 0.1 0.1 0.2 mg/L AST  30-300 141 75 11768 98 IU/L ALT  30-200 30 25 33 30 27 IU/L Albumin 2.5-4.0 2.9 2.9 2.92.9 2.9 g/dL Triglycerides  25-100* 213 271 280 296 271 mg/dLCholesterol  70-125 118 120 126 112 117 mg/dL Glucose  80-150* 155 162171 189 175 mg/dL*Triglyceride and glucose levels are routinely higher in the Balb/cstrain of mice than in other strains of mice.

The levels of routine clinical indicators of liver and kidney injury anddisease are within normal ranges and are not significantly changedrelative to saline-treated animals, demonstrating that the compounds ofthe invention do not significantly affect renal or hepatic function.Triglyceride and glucose levels, while outside the normal range as iscommon in the Balb/c strain, are not significantly elevated relative tosaline-treated animals.

The results illustrated in this example demonstrate that mixed backbonecompounds of varying chemistries are delivered to the kidney, reduceSGLT2 expression in vivo, and that treatment with these compounds doesnot result in liver or kidney toxicity. The results further indicatethat mixed backbone compounds with wings composed completely ofphosphodiester linkages are more efficient inhibitors of target mRNA.

Example 25 Effects of Modified Antisense Compounds on SGLT2 mRNAExpression in vivo: Two and Three Dose Protocols

In accordance with the present invention, mixed backbone compound ISIS257016 (SEQ ID NO; 106) was analyzed for its ability to inhibit SGLT2expression in vivo when administered in either two or three doses. ISIS353003 (CCTTCCCTGAAGGTTCCTCC; SEQ ID NO: 267), a mixed backboneoligonucleotide which targets human PTP1B, was used as a control.

Male 8-week old Balb/c mice (Charles River Laboratories, Wilmington,Mass.) were given two or three intraperitoneal injections of ISIS 257016or ISIS 353003 at three day intervals. ISIS 257016 was administered atdoses of 1, 5 or 25 mg/kg and ISIS 353003 was administered at a dose of25 mg/kg. Saline-injected animals served as controls. Each treatmentgroup contained 4 mice. The mice were sacrificed 2 days followingadministration of the final dose of oligonucleotide or saline.

Mice were evaluated for SGLT2 levels in kidney. Target levels weredetermined by quantitative real-time PCR as described in other examplesherein. PCR results were normalized to cyclophilin. The data areexpressed as percent change relative to saline treated animals (“+”indicates an increase, “−” indicates a decrease) and are illustrated inTable 31. TABLE 31 Antisense inhibition of SGLT2 mRNA expression in vivoby two doses or three doses of mixed backbone oligonucleotides(expressed as percent change in SGLT2 mRNA expression relative to salinecontrol) Oligonucleotide Three (dose in mg/kg) Two Doses Doses ISIS257016 (1 mg/kg) −43.2 −39.1 ISIS 257016 (5 mg/kg) −39.7 −42.9 ISIS257016 (25 mg/kg) −53.8 −65.5 ISIS 353003 (25 mg/kg) −8.0 −6.9

These results illustrate that the mixed backbone compounds of theinvention efficiently inhibit the expression of kidney SGLT2 in adose-dependent manner. Furthermore, inhibition increases with the numberof doses administered.

Treated mice were further evaluated for body weight, kidney weight,liver weight and spleen weight. The data are expressed as percent changein body or organ weight (“+” indicates an increase, “−” indicates adecrease). The results are presented in Table 32 and Table 33. TABLE 32Effects of antisense compounds on total body weight of mice (expressedas percent change in body weight) Oligonucleotide Two Three (dose inmg/kg) Doses Doses ISIS 257016 (1 mg/kg) −1.1 0 ISIS 257016 (5 mg/kg)+1.3 +0.8 ISIS 257016 (25 mg/kg) +0.1 +1.3 ISIS 353003 (25 mg/kg) −0.8+0.8

TABLE 33 Effects of antisense compounds on total kidney weight, liverweight and spleen weight of mice Percent change in weightOligonucleotide Two Doses Three Doses (dose in mg/kg) Kidney LiverSpleen Kidney Liver Spleen ISIS 257016 −0.5 −2.2 −4.3 −5.6 −3.8 −5.9 (1mg/kg) ISIS 257016 −5.4 +2.5 +7.4 −6.6 −7.1 −9.0 (5 mg/kg) ISIS 257016−7.9 −1.1 +4.2 −8.6 −8.8 −1.2 (25 mg/kg) ISIS 353003 −5.5 +1.2 −2.7 −0.2−4.0 +6.5 (25 mg/kg)

No significant change was observed in total body weight, kidney weight,liver weight or spleen weight at timepoints throughout or at thetermination of the study.

Levels of BUN, creatinine, bilirubin, AST, ALT, albumin, triglycerides,cholesterol and glucose were measured in mice treated with the compoundsof the invention. Plasma samples were analyzed using the Olympus AU400eautomated chemistry analyzer (Olympus America, Irving, Tex.). Theresults, expressed as units measured, are shown for the two doseprotocol in Table 34 and for the three dose protocol in Table 35. TABLE34 Effect of mixed backbone antisense compound ISIS 257016 administeredaccording to the two dose protocol on indicators of liver and kidneyfunction Units measured per dose of ISIS 257016 Serum Normal Sa- 1 5 25ISIS indicator Range line mg/kg mg/kg mg/kg 353003 BUN 15-40 32 34 29 2528 mg/dL Creatinine 0.0-1.0 0.1 0.1 0.2 0.1 0.1 mg/L Bilirubin 0.1-1.00.1 0.1 0.1 0.1 0.1 mg/dL AST  30-300 54 119 156 116 154 IU/L ALT 30-200 27 36 45 30 36 IU/L Albumin 2.5-4.0 2.7 3.2 3.1 3.0 2.8 g/dLTriglycerides  25-100* 221 263 234 264 278 mg/dL Cholesterol  70-125 113118 117 125 125 mg/dL Glucose  80-150* 170 157 177 163 152 mg/dL

TABLE 35 Effect of mixed backbone antisense compound ISIS 257016administered according to the three dose protocol on indicators of liverand kidney function Units measured per dose of ISIS 257016 Serum NormalSa- 1 5 25 ISIS indicator Range line mg/kg mg/kg mg/kg 353003 BUN 15-4030 32 30 27 27 mg/dL Creatinine 0.0-1.0 0.1 0.1 0.2 0.1 0.1 mg/LBilirubin 0.1-1.0 0.1 0.1 0.1 0.1 0.1 mg/dL AST  30-300 126 83 81 59 57IU/L ALT  30-200 35 30 57 27 24 IU/L Albumin 2.5-4.0 3.0 2.8 2.8 2.7 2.8g/dL Triglycerides  25-100* 223 236 202 153 188 mg/dL Cholesterol 70-125 112 113 114 116 106 mg/dL Glucose  80-150* 152 169 161 181 192mg/dL*Triglyceride and glucose levels are routinely higher in the Balb/cstrain of mice than in other strains of mice.

The levels of routine clinical indicators of liver and kidney injury anddisease are within normal ranges and are not significantly changedrelative to saline-treated animals, demonstrating that the compounds ofthe invention do not significantly affect renal or hepatic function.Triglyceride and glucose levels, while outside the normal range as iscommon in the Balb/c strain, are not significantly elevated relative tosaline-treated animals.

Mice injected with ISIS 257016 and control animals were also evaluatedhistologically following routine procedures. Liver and kidney sampleswere procured, fixed in 10% neutral-buffered formalin and processed forstaining with hematoxylin and eosin. Hematoxylin and eosin stainingexhibited no significant difference between saline- andoligonucleotide-treated animals. All tissue samples exhibited normalkidney and liver morphology.

The results illustrated in this example demonstrate that mixed backbonecompounds are delivered to the kidney, reduce SGLT2 expression in vivo,and that treatment with these compounds does not result in liver orkidney toxicity. The results further indicate that inhibition of targetmRNA expression in the kidney increases with the number of dosesadministered.

Example 26 Effects of Mixed Backbone Antisense Compounds on SGLT2 mRNAExpression in vivo: Two to Five Day Consecutive Daily Dosing Protocols

In accordance with the present invention, mixed backbone compound ISIS257016 (SEQ ID NO: 106) was analyzed for its ability to inhibit SGLT2expression in vivo when administered in two to five doses (consecutivedaily doses). ISIS 353003 (SEQ ID NO: 267), a mixed backboneoligonucleotide which targets human PTP IB, was used as a control.

Male 9-week old Balb/c mice (Charles River Laboratories, Wilmington,Mass.) were given two, three, four or five intraperitoneal injections ofISIS 257016 or ISIS 353003 once a day for the treatment period. ISIS257016 was administered at doses of 2.5 or 25 mg/kg and ISIS 353003 wasadministered at a dose of 25 mg/kg. Saline-injected animals served ascontrols. Each treatment group contained 4 mice. The mice weresacrificed 2 days following administration of the final dose ofoligonucleotide or saline.

Mice were evaluated for SGLT2 levels in kidney. Target levels weredetermined by quantitative real-time PCR as described in other examplesherein. PCR results were normalized to cyclophilin. The data areexpressed as percent change relative to saline treated animals (“+”indicates an increase, “−” indicates a decrease) and are illustrated inTable 36. TABLE 36 Antisense inhibition of SGLT2 mRNA expression in vivoby mixed backbone oligonucleotide (expressed as percent change in SGLT2mRNA expression relative to saline control) Oligonucleotide Three FourFive (dose in mg/kg) Two Doses Doses Doses Doses ISIS 257016 (2.5 mg/kg)−14.2 −35.4 −25.3 −42.0 ISIS 257016 (25 mg/kg) −12.5 −32.9 −39.1 −68.9ISIS 353003 (25 mg/kg) −4.5 −9.6 +0.5 −11.3

These results illustrate that the mixed backbone compounds of theinvention efficiently inhibit the expression of kidney SGLT2 andinhibition increases with the number of doses administered.

Treated mice were further evaluated for body weight, kidney weight,liver weight and spleen weight. The data are expressed as percent changein body or organ weight (“+” indicates an increase, “−” indicates adecrease). The results are presented in Tables 37-40. TABLE 37 Effectsof antisense compounds on total body weight of mice (expressed aspercent change in body weight) Oligonucleotide Two Three Four Five (dosein mg/kg) Doses Doses Doses Doses ISIS 257016 (2.5 mg/kg) +2.7 +2.7 +3.2+1.5 ISIS 257016 (25 mg/kg) +2.0 +2.0 +3.1 −0.7 ISIS 353003 (25 mg/kg)+0.6 +0.8 +2.5 +1.3

TABLE 38 Effects of antisense compounds on total kidney weight(expressed as percent change in kidney weight) Oligonucleotide Two ThreeFour Five (dose in mg/kg) Doses Doses Doses Doses ISIS 257016 (2.5mg/kg) +8.2 −1.4 +8.9 +1.5 ISIS 257016 (25 mg/kg) +11.5 +3.6 +2.7 −7.7ISIS 353003 (25 mg/kg) +5.3 −3.6 +4.9 +7.1

TABLE 39 Effects of antisense compounds on total liver weight (expressedas percent change in liver weight) Oligonucleotide Two Three Four Five(dose in mg/kg) Doses Doses Doses Doses ISIS 257016 (2.5 mg/kg) +9.2+7.5 +4.8 +4.8 ISIS 257016 (25 mg/kg) +11.8 +5.2 +0.6 −8.0 ISIS 353003(25 mg/kg) +7.4 −3.4 +12.9 +9.5

TABLE 40 Effects of antisense compounds on total spleen weight(expressed as percent change in spleen weight) Oligonucleotide Two ThreeFour Five (dose in mg/kg) Doses Doses Doses Doses ISIS 257016 (2.5mg/kg) +22.2 +10.1 +15.3 +10.7 ISIS 257016 (25 mg/kg) +13.3 +5.1 +6.7+4.5 ISIS 353003 (25 mg/kg +7.3 +1.4 +19.8 +8.6

No significant change was observed in total body weight, kidney weight,liver weight or spleen weight at timepoints throughout or at thetermination of the study.

Levels of creatinine, AST, ALT, albumin, triglycerides, cholesterol andglucose were measured in mice treated with the compounds of theinvention. Plasma samples were analyzed using the Olympus AU400eautomated chemistry analyzer (Olympus America, Irving, Tex.). Theresults, expressed as units measured, are shown in Tables 41-44. TABLE41 Effect of mixed backbone antisense compound ISIS 257016 administeredas two consecutive daily doses on indicators of liver and kidneyfunction Units measured per dose of oligonucleotide ISIS ISIS ISIS SerumNormal 257016 257016 353003 indicator Range Saline 2.5 mg/kg 25 mg/kg 25mg/kg Creatinine 0.0-1.0 0.2 0.1 0.1 0.2 mg/L AST  30-300 160 132 75 131IU/L ALT  30-200 31 31 28 29 IU/L Albumin 2.5-4.0 2.8 2.9 3.0 2.7 g/dLTriglycerides  25-100* 233 238 287 240 mg/dL Cholesterol  70-125 113 117122 106 mg/dL Glucose  80-150* 171 195 175 164 mg/dL

TABLE 42 Effect of mixed backbone antisense compound ISIS 257016administered as three consecutive daily doses on indicators of liver andkidney function Units measured per dose of oligonucleotide ISIS ISISISIS Serum Normal 257016 257016 353003 indicator Range Saline 2.5 mg/kg25 mg/kg 25 mg/kg Creatinine 0.0-1.0 0.1 0.2 0.2 0.1 mg/L AST  30-300199 60 84 155 IU/L ALT  30-200 29 28 26 77 IU/L Albumin 2.5-4.0 2.8 2.92.8 2.9 g/dL Triglycerides  25-100* 289 268 259 236 mg/dL Cholesterol 70-125 111 118 108 106 mg/dL Glucose  80-150* 204 162 181 179 mg/dL

TABLE 43 Effect of mixed backbone antisense compound ISIS 257016administered as four consecutive daily doses on indicators of liver andkidney function Units measured per dose of oligonucleotide ISIS ISISISIS Serum Normal 257016 257016 353003 indicator Range Saline 2.5 mg/kg25 mg/kg 25 mg/kg Creatinine 0.0-1.0 0.1 0.1 0.1 0.2 mg/L AST  30-300199 92 120 144 IU/L ALT  30-200 29 30 30 36 IU/L Albumin 2.5-4.0 2.8 3.02.8 3.0 g/dL Triglycerides  25-100* 289 252 269 294 mg/dL Cholesterol 70-125 111 126 115 120 mg/dL Glucose  80-150* 204 173 198 192 mg/dL

TABLE 44 Effect of mixed backbone antisense compound ISIS 257016administered as five consecutive daily doses on indicators of liver andkidney function Units measured per dose of oligonucleotide ISIS ISISISIS Serum Normal 257016 257016 353003 indicator Range Saline 2.5 mg/kg25 mg/kg 25 mg/kg Creatinine 0.0-1.0 0.1 0.1 0.1 0.1 mg/L AST  30-300129 121 125 97 IU/L ALT  30-200 30 30 33 29 IU/L Albumin 2.5-4.0 2.8 2.92.8 2.9 g/dL Triglycerides  25-100* 298 298 285 277 mg/dL Cholesterol 70-125 116 126 122 126 mg/dL Glucose  80-150* 163 177 204 185 mg/dL*Triglyceride and glucose levels are routinely higher in the Balb/cstrain of mice than in other strains of mice.

The levels of routine clinical indicators of liver and kidney injury anddisease are within normal ranges and are not significantly changedrelative to saline-treated animals, demonstrating that the compounds ofthe invention do not significantly affect renal or hepatic function.Triglyceride and glucose levels, while outside the normal range as iscommon in the Balb/c strain, are not significantly elevated relative tosaline-treated animals.

The results illustrated in this example demonstrate that mixed backbonecompounds are delivered to the kidney, reduce SGLT2 expression in vivo,and that treatment with these compounds does not result in liver orkidney toxicity. The results further indicate that inhibition of targetmRNA expression in the kidney increases with the number of dosesadministered.

Example 27 Comparison of a Standard Mixed Backbone Compound and MixedBackbone Compounds with Phosphorothioate Linkages at Either or Both ofthe Extreme 5′ and 3′ Ends: a Four Dose Protocol

In accordance with the present invention, ISIS 257016 (SEQ ID NO: 106),ISIS 351641 (SEQ ID NO: 106), ISIS 360886 (SEQ ID NO: 106) and ISIS360887 (SEQ ID NO: 106) were analyzed for their ability to inhibit SGLT2expression in vivo. ISIS 257016 is a standard mixed backbone compoundhaving 2′-MOE wings and a deoxy gap, with phosphodiester linkages in thewings and phosphorothioate linkages in the gap. ISIS 351641 differs fromthe standard mixed backbone compounds by having one phosphorothioatelinkage at each of the extreme 5′ and 3′ ends of the wings. ISIS 360886and ISIS 360887 are mixed backbone compounds with one phosphorothioatelinkage at the extreme 5′ end or extreme 3′ end, respectively.

Male 7-week old Balb/c mice (Charles River Laboratories, Wilmington,Mass.) were given intraperitoneal injections of ISIS 257016, ISIS351641, ISIS 360886 or ISIS 360887 twice a week for two weeks at dosesof 1.56, 6.25 or 25 mg/kg. Saline-injected animals served as controls.Each treatment group contained 4 mice. The mice were sacrificed 2 daysfollowing administration of the final dose of oligonucleotide or saline.

Mice were evaluated for SGLT2 levels in kidney. Target levels weredetermined by quantitative real-time PCR as described in other examplesherein. PCR results were normalized to cyclophilin. The data areexpressed as percent change relative to saline treated animals (“+”indicates an increase, “−” indicates a decrease) and are illustrated inTable 45. TABLE 45 Antisense inhibition of SGLT2 mRNA expression in vivoby mixed backbone oligonucleotides (expressed as percent change in SGLT2mRNA expression relative to saline) Dose of oligonucleotide ISIS ISISISIS ISIS mg/kg 257016 351641 360886 360887 1.56 −39.1 −4.2 −12.7 −9.76.25 −52.8 −4.87 −19.7 −7.3 25 −57.8 −11.0 −29.0 −4.9

These results illustrate that mixed backbone compounds of the invention,with either complete phosphodiester linkages in the wings, or with theextreme 5′ and 3′ ends substituted with phosphorothioate linkages, caninhibit the expression of kidney SGLT2 in a dose-dependent manner. Withthe exception of ISIS 360887, inhibition of target mRNA wasdose-dependent. Although all mixed backbone compounds inhibited SGLT2expression, greater inhibition is observed in kidneys from mice treatedwith ISIS 257016, which is a mixed backbone compound that contains allphosphodiester linkages in the wings.

Treated mice were further evaluated for body weight, kidney weight,liver weight and spleen weight. The data are expressed as percent changein body or organ weight (“+” indicates an increase, “−” indicates adecrease). The results are presented in Table 46. TABLE 46 Effects ofantisense compounds on total body weight, kidney weight, liver weightand spleen weight of mice (expressed as percent change in weight) DoseBody Kidney Liver Spleen Oligonucleotide mg/kg weight weight weightweight ISIS 257016 1.56 +11.6 −3.5 −4.2 −2.4 ISIS 257016 6.25 +7.9 −3.0+3.8 −1.3 ISIS 257016 25 +11.7 −4.1 +1.4 +8.9 ISIS 351641 1.56 +7.9 −0.9−5.4 +9.4 ISIS 351641 6.25 +11.1 +1.3 −2.2 +13.4 ISIS 351641 25 +7.4−2.1 −0.5 −1.4 ISIS 360886 1.56 +7.6 −1.0 −13.7 −5.0 ISIS 360886 6.25+8.9 −3.7 −16.6 +1.2 ISIS 360886 25 +11.1 −5.5 −11.6 +0.8 ISIS 3608871.56 +8.5 +1.0 −10.4 −0.4 ISIS 360887 6.25 +7.5 −1.8 −8.4 +1.1 ISIS360887 25 +9.8 +2.2 −9.0 +11.8

No significant change was observed in total body weight, liver weight orspleen weight at timepoints throughout or at the termination of thestudy.

Levels of BUN, creatinine, bilirubin, AST, ALT, albumin, triglycerides,cholesterol and glucose were measured in mice treated with the compoundsof the invention. Plasma samples were analyzed using the Olympus AU400eautomated chemistry analyzer (Olympus America, Irving, Tex.). Theresults, expressed as units measured, are shown in Tables 47-50. TABLE47 Effect of mixed backbone antisense compound ISIS 257016 on indicatorsof liver and kidney function Units measured per dose of ISIS 257016Serum Normal 1.56 6.25 25 indicator Range Saline mg/kg mg/kg mg/kg BUN 15-40 23 21 26 22 mg/dL Creatinine 0.0-1.0 0.2 0.2 0.2 0.2 mg/LBilirubin mg/dL 0.1-1.0 0.2 0.2 0.2 0.1 AST  30-300 75 61 83 71 IU/L ALT 30-200 30 30 33 39 IU/L Albumin 2.5-4.0 2.8 2.9 2.9 2.7 g/dLTriglycerides  25-100* 208 210 243 150 mg/dL Cholesterol  70-125 116 125130 135 mg/dL Glucose  80-150* 207 184 184 215 mg/dL

TABLE 48 Effect of mixed backbone antisense compound ISIS 351641 onindicators of liver and kidney function Units measured per dose of ISIS351641 Normal 1.56 6.25 25 Serum indicator Range Saline mg/kg mg/kgmg/kg BUN  15-40 23 23 25 22 mg/dL Creatinine 0.0-1.0 0.2 0.2 0.2 0.2mg/L Bilirubin mg/dL 0.1-1.0 0.2 0.1 0.2 0.1 AST  30-300 75 61 67 54IU/L ALT  30-200 30 32 31 30 IU/L Albumin 2.5-4.0 2.8 2.7 2.7 2.8 g/dLTriglycerides  25-100* 208 169 176 185 mg/dL Cholesterol  70-125 116 110115 107 mg/dL Glucose  80-150* 207 205 199 208 mg/dL

TABLE 49 Effect of mixed backbone antisense compound ISIS 360886 onindicators of liver and kidney function Units measured per dose of ISIS360886 Normal 1.56 6.25 25 Serum indicator Range Saline mg/kg mg/kgmg/kg BUN  15-40 23 21 23 24 mg/dL Creatinine 0.0-1.0 0.2 0.1 0.2 0.2mg/L Bilirubin mg/dL 0.1-1.0 0.2 0.2 0.2 0.1 AST  30-300 75 56 77 73IU/L ALT  30-200 30 26 27 28 IU/L Albumin 2.5-4.0 2.8 2.7 2.7 2.7 g/dLTriglycerides  25-100* 208 164 181 169 mg/dL Cholesterol  70-125 116 105108 108 mg/dL Glucose  80-150* 207 189 202 200 mg/dL

TABLE 50 Effect of mixed backbone antisense compound ISIS 360887 onindicators of liver and kidney function Units measured per dose of ISIS360887 Normal 1.56 6.25 25 Serum indicator Range Saline mg/kg mg/kgmg/kg BUN  15-40 23 23 22 23 mg/dL Creatinine 0.0-1.0 0.2 0.2 0.2 0.2mg/L Bilirubin mg/dL 0.1-1.0 0.2 0.2 0.1 0.2 AST  30-300 75 142 83 108IU/L ALT  30-200 30 40 39 34 IU/L Albumin 2.5-4.0 2.8 2.7 2.7 2.7 g/dLTriglycerides  25-100* 208 136 157 200 mg/dL Cholesterol  70-125 116 109107 110 mg/dL Glucose  80-150* 207 199 201 187 mg/dL*Triglyceride and glucose levels are routinely higher in the Balb/cstrain of mice than in other strains of mice.

Cholesterol levels of mice treated with either 6.25 or 25 mg/kg wereslightly elevated; however, these levels are not significantly greaterthan the cholesterol levels observed in saline-treated control animals.Otherwise, the levels of routine clinical indicators of liver and kidneyinjury and disease are within normal ranges and are not significantlychanged relative to saline-treated animals, demonstrating that thecompounds of the invention do not significantly affect renal or hepaticfunction. Triglyceride and glucose levels, while outside the normalrange as is common in the Balb/c strain, are not significantly elevatedrelative to saline-treated animals.

Saline- and oligonucleotide-injected animals also were evaluatedhistologically following routine procedures. Liver and kidney sampleswere procured, fixed in 10% neutral-buffered formalin and processed forstaining with hematoxylin and eosin. Hematoxylin and eosin stainingexhibited no significant difference between control andoligonucleotide-treated animals.

The results illustrated in this example demonstrate that mixed backbonecompounds are delivered to the kidney, reduce SGLT2 expression in vivo,and that treatment with these compounds does not result in liver orkidney toxicity. The results further indicate that mixed backbonecompounds with complete phosphodiester linkages in the wings are moreeffective modulators of target mRNA expression in the kidney than mixedbackbone compounds with a phosphorothioate linkage at one or both of theextreme 5′ and 3′ ends.

Example 28 Comparison of a Standard Mixed Backbone Compound and MixedBackbone Compounds with Phosphorothioate Linkages at Either or Both ofthe Extreme 5′ and 3′ Ends: an Eight Dose Protocol

A second study of SGLT2 antisense oligonucleotides ISIS 257016, ISIS351641, ISIS 360886 and ISIS 360887 was undertaken in which micereceived eight doses over a four week period. As described previously,ISIS 257016 is a standard mixed backbone compound having 2′-MOE wingsand a deoxy gap, with phosphodiester linkages in the wings andphosphorothioate linkages in the gap. ISIS 351641 differs from thestandard mixed backbone compounds by having one phosphorothioate linkageat each of the extreme 5′ and 3′ ends of the wings. ISIS 360886 and ISIS360887 are mixed backbone compounds with one phosphorothioate linkage atthe extreme 5′ end and extreme 3′ end, respectively.

Male 8-week old Balb/c mice (Charles River Laboratories, Wilmington,Mass.) were given intraperitoneal injections of ISIS 257016, ISIS351641, ISIS 360886 or ISIS 360887 twice a week for four weeks at dosesof 1, 5 or 25 mg/kg. Saline-injected animals served as controls. Eachtreatment group contained 4 mice. The mice were sacrificed 2 daysfollowing administration of the final dose of oligonucleotide or saline.

Mice were evaluated for SGLT2 levels in kidney. Target levels weredetermined by quantitative real-time PCR as described by other examplesherein. PCR results were normalized to cyclophilin. The data areexpressed as percent change relative to saline treated animals (“+”indicates an increase, “−” indicates a decrease) and are illustrated inTable 51. TABLE 51 Antisense inhibition of SGLT2 mRNA expression in vivoby mixed backbone oligonucleotides (expressed as percent change in SGLT2mRNA expression relative to saline) Dose of oligonucleotide ISIS ISISISIS ISIS mg/kg 257016 351641 360886 360887 1 −53 −14 −24 −23 5 −64 −23−30 −26 25 −68 −37 −50 −40

These results illustrate that mixed backbone compounds of the invention,with either complete phosphodiester linkages in the wings, or with theextreme 5′ and 3′ ends substituted with phosphorothioate linkages, caninhibit the expression of kidney SGLT2 in a dose-dependent manner.However, greater inhibition is observed in kidneys from mice treatedwith ISIS 257016, which contains all phosphodiester linkages in thewings.

Treated mice were further evaluated for body weight and liver and spleenweight. The data are expressed as percent change in body or organ weight(“+” indicates an increase, “−” indicates a decrease). The results arepresented in Table 52. TABLE 52 Effects of antisense compounds on totalbody weight, liver weight and spleen weight of mice (expressed aspercent change in weight) Dose Body Liver Spleen Oligonucleotide mg/kgweight weight weight ISIS 257016 1 +11.8 −6.9 −10.1 ISIS 257016 5 +8.4−4.3 +4.4 ISIS 257016 25 +5.4 −2.1 +12.5 ISIS 351641 1 +12.3 −2.8 −2.9ISIS 351641 5 +9.2 −8.7 −5.5 ISIS 351641 25 +9.4 −0.8 +3.3 ISIS 360886 1+9.2 −5.2 −4.5 ISIS 360886 5 +10.3 −2.7 +15.1 ISIS 360886 25 +9.4 −2.1−11.4 ISIS 360887 1 +10.0 −7.0 −1.5 ISIS 360887 5 +12.6 −3.2 +4.0 ISIS360887 25 +11.8 −7.6 +14.7

No significant change was observed in total body weight, liver weight orspleen weight at timepoints throughout or at the termination of thestudy.

Levels of BUN, creatinine, bilirubin, AST, ALT, albumin, triglycerides,cholesterol and glucose were measured in mice treated with the compoundsof the invention. Plasma samples were analyzed using the Olympus AU400eautomated chemistry analyzer (Olympus America, Irving, Tex.). Theresults, expressed as units measured, are shown in Tables 53-56. TABLE53 Effect of mixed backbone antisense compound ISIS 257016 on indicatorsof liver and kidney function Units measured per Normal dose of ISIS257016 Serum indicator Range Saline 1 mg/kg 5 mg/kg 25 mg/kg BUN  15-4027 31 29 23 mg/dL Creatinine 0.0-1.0 0.2 0.2 0.2 0.2 mg/L Bilirubinmg/dL 0.1-1.0 0.2 0.2 0.2 0.2 AST  30-300 60 58 82 119 IU/L ALT  30-20022 27 35 66 IU/L Albumin 2.5-4.0 2.7 2.8 2.7 2.6 g/dL Triglycerides 25-100* 178 263 187 99 mg/dL Cholesterol  70-125 123 142 138 162 mg/dLGlucose  80-150* 193 201 201 185 mg/dL

TABLE 54 Effect of mixed backbone antisense compound ISIS 351641 onindicators of liver and kidney function Units measured per Normal doseof ISIS 351641 Serum indicator Range Saline 1 mg/kg 5 mg/kg 25 mg/kg BUN15-40 27 27 26 28 mg/dL Creatinine 0.0-1.0 0.2 0.2 0.2 0.2 mg/LBilirubin 0.1-1.0 0.2 0.1 0 0.1 mg/dL AST  30-300 60 48 49 50 IU/L ALT 30-200 22 23 23 20 IU/L Albumin 2.5-4.0 2.7 2.8 2.8 2.7 g/dLTriglycerides  25-100* 178 165 197 222 mg/dL Cholesterol  70-125 123 118120 118 mg/dL Glucose  80-150* 193 192 200 197 mg/dL

TABLE 55 Effect of mixed backbone antisense compound ISIS 360886 onindicators of liver and kidney function Units measured per Normal doseof ISIS 360886 Serum indicator Range Saline 1 mg/kg 5 mg/kg 25 mg/kg BUN15-40 27 27 26 27 mg/dL Creatinine 0.0-1.0 0.2 0.2 0.2 0.2 mg/LBilirubin 0.1-1.0 0.2 0 0.1 0.1 mg/dL AST  30-300 60 52 71 90 IU/L ALT 30-200 22 23 23 29 IU/L Albumin 2.5-4.0 2.7 2.8 2.8 2.8 g/dLTriglycerides  25-100* 178 230 250 227 mg/dL Cholesterol  70-125 123 122129 133 mg/dL Glucose  80-150* 193 187 182 185 mg/dL

TABLE 56 Effect of mixed backbone antisense compound ISIS 360887 onindicators of liver and kidney function Units measured per Normal doseof ISIS 360887 Serum indicator Range Saline 1 mg/kg 5 mg/kg 25 mg/kg BUN15-40 27 25 24 23 mg/dL Creatinine 0.0-1.0 0.2 0.2 0.2 0.1 mg/LBilirubin 0.1-1.0 0.2 0.2 0.2 0.2 mg/dL AST  30-300 60 60 44 92 IU/L ALT 30-200 22 24 22 31 IU/L Albumin 2.5-4.0 2.7 2.7 2.5 2.7 g/dLTriglycerides  25-100* 178 240 262 171 mg/dL Cholesterol  70-125 123 121129 134 mg/dL Glucose  80-150* 193 189 186 181 mg/dL*Triglyceride and glucose levels are routinely higher in the Balb/cstrain of mice than in other strains of mice.

The levels of routine clinical indicators of liver and kidney injury anddisease are within normal ranges and are not significantly changedrelative to saline-treated animals, demonstrating that the compounds ofthe invention do not significantly affect renal or hepatic function.Triglyceride and glucose levels, while outside the normal range as iscommon in the Balb/c strain, are not significantly elevated relative tosaline-treated animals.

The results illustrated in this example demonstrate that mixed backbonecompounds are delivered to the kidney, reduce SGLT2 expression in vivo,and that treatment with these compounds does not result in liver orkidney toxicity. Furthermore, the eight dose protocol resulted ingreater inhibition of target mRNA levels in the kidney than observed forthe four dose protocol shown in Example 22.

Example 29 Antisense Inhibition of SGLT2 in a Murine Model of Type 2Diabetes: Comparison of Full Phosphorothioate and Mixed BackboneOligonucleotides

The Animal Models of Diabetic Complications Consortium (AMDCC) hasdeveloped protocols for the induction of diabetes in a number of animalmodels. The genetic C57BLKS/J Lep^(db)/Lep^(db) model has been approvedby the AMDCC as an appropriate model system for studies of diabeticnephropathy associated with type 2 diabetes.

Leptin is a hormone produced by fat that regulates appetite.Deficiencies in this hormone in both humans and non-human animals leadto obesity. Lep^(db)/Lep^(db) mice have a mutation in the leptinreceptor gene which results in obesity and hyperglycemia. As such, thesemice are a useful model for the investigation of obesity and diabetesand treatments designed to treat these conditions. In accordance withthe present invention, oligomeric compounds of the present inventionwere tested in the Lep^(db)/Lep^(db) model of type 2 diabetes.

Male Lep^(db)/Lep^(db) (db/db) mice were given intraperitonealinjections of either ISIS 257016 (SEQ ID NO: 106), which has a mixedbackbone, or ISIS 145733 (SEQ ID NO: 106), which has a phosphorothioatebackbone, twice a week for four weeks at doses of 12.5, 25 or 37.5mg/kg. Saline-injected animals served as controls. Each treatment groupcontained 6 mice. The mice were sacrificed 2 days followingadministration of the final dose of oligonucleotide or saline.

Mice were evaluated for SGLT2 levels in kidney. Target levels weredetermined by quantitative real-time PCR as described by other examplesherein. PCR results were normalized to cyclophilin. The data areexpressed as percent change relative to saline treated animals (“+”indicates an increase, “−” indicates a decrease) and are illustrated inTable 57. TABLE 57 Antisense inhibition of SGLT2 mRNA expression indb/db mice (expressed as percent change in SGLT2 mRNA expressionrelative to saline) Dose of oligonucleotide ISIS ISIS mg/kg 145733257016 12.5 −48 −72 25 −71 −72 37.5 −64 −72

These results illustrate that both mixed backbone compound ISIS 257016and full phosphorothioate compound ISIS 145733 effectively inhibit theexpression of kidney SGLT2. However, greater inhibition is observed inkidneys from mice treated with ISIS 257016, particularly at the lowestdose of 12.5 mg/kg.

Treated mice were further evaluated for body weight and liver and spleenweight. The data are expressed as weight in grams. The results arepresented in Table 58. TABLE 58 Effects of antisense compounds on totalbody weight, liver weight and spleen weight of db/db mice (in grams)Dose Body Kidney Liver Spleen Oligonucleotide mg/kg weight weight weightweight Saline — 35 0.32 1.5 0.09 ISIS 145733 12.5 34 0.32 1.9 0.12 ISIS145733 25 37 0.37 2.1 0.15 ISIS 145733 37.5 38 0.35 2.3 0.14 ISIS 25701612.5 34 0.31 1.6 0.09 ISIS 257016 25 36 0.31 1.7 0.08 ISIS 257016 37.534 0.35 1.8 0.11

No significant change was observed in total body weight, liver weight orspleen weight at timepoints throughout or at the termination of thestudy.

Levels of AST, ALT, triglycerides, cholesterol and glucose were measuredin mice treated with the compounds of the invention. Plasma samples wereanalyzed using the Olympus AU400e automated chemistry analyzer (OlympusAmerica, Irving, Tex.). The results, expressed as units measured, areshown in Table 59 and Table 60. TABLE 59 Effect of full phosphorothioatebackbone compound ISIS 145733 on indicators of toxicity Units measuredper dose of ISIS 145733 Normal 12.5 mg/ 25 mg/ 37.5 mg/ Serum indicaterRange Saline kg kg kg AST 30-300  61 72 80 93 IU/L ALT 30-200  63 87 101120 IU/L Triglycerides 25-100* 245 216 243 204 mg/dL Cholesterol 70-125*182 196 211 224 mg/dL Glucose 80-150* 611 452 391 351 mg/dL

TABLE 60 Effect of mixed backbone antisense compound ISIS 257016 onindicators of toxicity Units measured per dose of ISIS 257016 Normal12.5 mg/ 25 mg/ 37.5 mg/ Serum indicater Range Saline kg kg kg AST30-300  61 120 144 175 IU/L ALT 30-200  63 123 142 154 IU/LTriglycerides 25-100* 245 167 188 183 mg/dL Cholesterol 70-125* 182 248264 265 mg/dL Glucose 80-150* 611 281 320 326 mg/dL*Triglyceride, cholesterol and glucose levels are routinely higher inthe Lep^(db)/Lep^(db) strain of mice than in other strains of mice.

The levels of routine clinical indicators of liver injury and diseaseare within normal ranges and are not significantly changed relative tosaline-treated animals, demonstrating that the compounds of theinvention do not significantly affect hepatic function. Given thegenetic defect of the Lep^(db)/Lep^(db) mice and the diabetic phenotypeexhibited by these mice, it is expected that triglyceride, cholesteroland glucose levels will exceed the normal range. Importantly, treatmentwith either of the SGLT2 antisense compounds resulted in a significantdecrease in blood glucose levels, with ISIS 257016, the mixed backbonecompound, achieving greater levels of target mRNA inhibition. Treatmentwith ISIS 257016 also resulted in a significant decrease in serumtriglyceride levels.

The results illustrated in this example demonstrate that mixed backbonecompounds are effectively delivered to the kidney, reduce SGLT2expression in vivo, and that treatment with these compounds does notresult in liver or other toxicity. Furthermore, these results indicatethat mixed backbone compounds targeted to SGLT2 efficiently decreaseblood glucose levels and serum triglyceride levels in a mouse model oftype 2 diabetes.

Example 30 Antisense Inhibition of SGLT2 in a Murine Model of Type 2Diabetes: Low Dose Comparison of Full Phosphorothioate and MixedBackbone Oligonucleotides

Since treatment with ISIS 257016 resulted in significant reduction inSGLT2 expression levels even at the lowest dose of 12.5 mg/kg, a seconddose-response study was conducted using a lower dose range of 1.56, 3.12and 6.25 mg/kg. Male Lep^(db)/Lep^(db) mice were given intraperitonealinjections of either mixed backbone compound ISIS 257016 or fullphosphorothioate compound ISIS 145733 twice a week for four weeks atdoses of 1.56, 3.12 or 6.25 mg/kg. Saline-injected animals served ascontrols. Each treatment group contained 4 mice. The mice weresacrificed 2 days following administration of the final dose ofoligonucleotide or saline.

Mice were evaluated for SGLT2 levels in kidney. Target levels weredetermined by quantitative real-time PCR as described by other examplesherein. PCR results were normalized to cyclophilin. The data areexpressed as percent change relative to saline treated animals (“+”indicates an increase, “−” indicates a decrease) and are illustrated inTable 61. TABLE 61 Antisense inhibition of SGLT2 mRNA expression indb/db mice (expressed as percent change in SGLT2 mRNA expressionrelative to saline) Dose of oligonucleotide ISIS ISIS mg/kg 145733257016 1.56 −13 −75 3.12 −14 −83 6.25 −12 −80

These results illustrate that mixed backbone compound ISIS 257016 is amore effective inhibitor of SGLT2 mRNA expression in the kidney,particularly at low doses of oligonucleotide.

Levels of glucose were measured in mice treated with the compounds ofthe invention. Plasma samples were analyzed using the Olympus AU400eautomated chemistry analyzer (Olympus America, Irving, Tex.). The dataare expressed as percent change relative to saline treated animals (“+”indicates an increase, “−” indicates a decrease) and are illustrated inTable 62. TABLE 62 Blood glucose levels in db/db mice treated with SGLT2antisense compounds (expressed as percent change in blood glucoserelative to saline) Dose of oligonucleotide ISIS ISIS mg/kg 145733257016 1.56 −5 −41 3.12 −7 −37 6.25 −14 −40

The results demonstrate that treatment with mixed backbone compound ISIS257016 results in a significant decrease in blood glucose levels andthat mixed backbone compounds are more effective at lowering bloodglucose levels than full phosphorothioate antisense compounds.

Antisense inhibition of SGLT2 by ISIS 257016 was further evaluated usinga dose range of 0.39, 0.78 and 1.56 mg/kg. As described above, maleLep^(db)/Lep^(db) mice were given intraperitoneal injections of mixedbackbone compound ISIS 257016 twice a week for four weeks.Saline-injected animals served as controls. Each treatment groupcontained 4 mice. The mice were sacrificed 2 days followingadministration of the final dose of oligonucleotide or saline.

Mice were evaluated for SGLT2 levels in kidney. Target levels weredetermined by quantitative real-time PCR as described by other examplesherein. PCR results were normalized to cyclophilin. Blood glucose levelsalso were determined. Plasma samples were analyzed using the OlympusAU400e automated chemistry analyzer (Olympus America, Irving, Tex.). Thedata are expressed as percent change relative to saline treated animals(“+” indicates an increase, “−′ indicates a decrease) and areillustrated in Table 63. TABLE 63 Antisense inhibition of SGLT2 mRNAexpression and blood glucose levels in db/db mice (expressed as percentchange in SGLT2 mRNA expression or blood glucose levels relative tosaline) Dose of ISIS 257016 SGLT2 Blood mg/kg mRNA glucose 0.39 −66 −160.78 −68 −21 1.56 −82 −21

These results further demonstrate the effectiveness of mixed backbonecompounds at inhibiting SGLT2 expression in the kidney and loweringblood glucose levels when administered at very low doses ofoligonucleotide.

Mice treated with the compounds of the invention also were evaluated forliver and kidney toxicity, organ and body weights and tissue histology.These studies demonstrated no significant level of toxicity or change inbody or organ weight, indicating that mixed backbone compounds areeffective in vivo without toxicity to the animal.

The results illustrated in this example demonstrate that mixed backbonecompounds are effectively delivered to the kidney, reduce SGLT2expression in vivo, and that treatment with these compounds lowers bloodglucose levels in diabetic animals.

Example 31 Antisense Inhibition of SGLT2 in a Murine Model of Obesityand Diabetes Using Mixed Backbone Compounds

Leptin is a hormone produced by fat that regulates appetite.Deficiencies in this hormone in both humans and non-human animals leadsto obesity. C57B1/6J-Lep ob/ob mice have a mutation in the leptin genewhich results in obesity and hyperglycemia. As such, these mice are auseful model for the investigation of obesity and diabetes andtreatments designed to treat these conditions. In accordance with thepresent invention, the oligomeric compounds of the invention were testedin the ob/ob model of obesity and diabetes.

Male C57B1/6J-Lep ob/ob mice (Jackson Laboratory, Bar Harbor, Me.) weresubcutaneously injected with ISIS 257016 (SEQ ID NO: 106) at a dose of25 mg/kg two times per week for 4 weeks. Saline-injected animals servedas controls. Each treatment group contained 4 mice. The mice weresacrificed 2 days following administration of the final dose ofoligonucleotide or saline.

Mice were evaluated for SGLT2 levels in kidney. Target levels weredetermined by quantitative real-time PCR as described by other examplesherein. PCR results were normalized to cyclophilin. Blood glucose levelsalso were determined. Plasma samples were analyzed using the OlympusAU400e automated chemistry analyzer (Olympus America, Irving, Tex.). Thedata are expressed as percent change relative to saline treated animals(“+” indicates an increase, “−” indicates a decrease) and areillustrated in Table 64. TABLE 64 Antisense inhibition of SGLT2 mRNAexpression and blood glucose levels in ob/ob mice (expressed as percentchange in SGLT2 mRNA expression or blood glucose levels relative tosaline) Dose of oligonucleotide SGLT2 Blood mg/kg mRNA glucose 25 −83−39

The results demonstrate that treatment with a mixed backbone SGLT2antisense compound results in a significant decrease in SGLT2 mRNAexpression in the kidney of diabetic mice. Importantly, blood glucoselevels also are significantly decreased in treated animals.

Example 32 Comparison of Mixed Backbone Compounds 16 to 20 Nucleobasesin Length

In accordance with the present invention, mixed backbone compounds withless than 20 nucleobases were evaluated for their ability to inhibitSGLT2 expression in the kidney. Four compounds were synthesized based onthe sequence of ISIS 257016 (SEQ ID NO: 106). ISIS 366847, ISIS 366848,ISIS 366849 and ISIS 366850 are comprised of the 5′-most 19, 18, 17 and16 nucleobases, respectively, of ISIS 257016 (see Table 65). ISIS 257016has 2′-MOE wings of five nucleobases each and a deoxy gap of 10nucleobases. ISIS 366847, ISIS 366848, ISIS 366849 and ISIS 366850 havea 10 nucleobases gap, a five nucleobase 2′-MOE wing at the 5′ end, butcontain a shortened 3′ wing of 1 to 4 nucleobases. TABLE 65 Antisensecompounds 16 to 20 nucleobases in length ISIS # SEQUENCE SEQ ID NO:257016 GAAGTAGCCACCAACTGTGC 106 366847 GAAGTAGCCACCAACTGTG 366848GAAGTAGCCACCAACTGT 366849 GAAGTAGCCACCAACTG 366850 GAAGTAGCCACCAACT

Male 6-week old Balb/c mice (Charles River Laboratories, Wilmington,Mass.) were given intraperitoneal injections of ISIS 257016, ISIS366847, ISIS 366848, ISIS 366849 or ISIS 366850 twice a week for twoweeks at doses of 0.14, 0.7 or 3.5 micromoles per kilogram (EM/kg).Saline-injected animals served as controls. Each treatment groupcontained 4 mice. The mice were sacrificed 2 days followingadministration of the final dose of oligonucleotide or saline.

Mice were evaluated for SGLT2 levels in kidney. Target levels weredetermined by quantitative real-time PCR as described in other examplesherein. PCR results were normalized to cyclophilin. The data areexpressed as percent change relative to saline treated animals (“+”indicates an increase, “−” indicates a decrease) and are illustrated inTable 66. TABLE 66 Antisense inhibition of SGLT2 mRNA expression in vivoby mixed backbone oligonucleotides (expressed as percent change relativeto saline control) Dose of oligonucleotide ISIS ISIS ISIS ISIS ISISμM/kg 257016 366847 366848 366849 366850 0.14 −53 −55 −58 −57 −49 0.7−56 −63 −59 −61 −57 3.5 −70 −64 −72 −69 −69

These results illustrate that mixed backbone compounds of the invention,containing 16 to 20 nucleobases, are effective inhibitors of SGLT2expression in the kidney.

Treated mice were further evaluated for body weight, kidney weight,liver weight and spleen weight. The data are expressed as percent changein body or organ weight (“+” indicates an increase, “−” indicates adecrease). The results are presented in Table 67. TABLE 67 Effects ofantisense compounds on total body weight, kidney weight, liver weightand spleen weight of mice (expressed as percent change in weight) DoseBody Kidney Liver Spleen Oligonucleotide μM/kg weight weight weightweight ISIS 257016 0.14 +9.0 −4.5 −6.1 −8.3 ISIS 257016 0.7 +11.1 −5.3+4.1 −3.7 ISIS 257016 3.5 +10.2 −3.6 +3.7 +11.9 ISIS 366847 0.14 +15.0−0.5 +0.2 −6.9 ISIS 366847 0.7 +12.7 +1.2 +6.8 −4.9 ISIS 366847 3.5+10.3 +3.6 +3.8 +2.9 ISIS 366848 0.17 +8.5 −7.1 −7.9 −2.4 ISIS 3668480.7 +7.7 +6.4 +5.9 +3.8 ISIS 366848 3.5 +10.8 +3.0 +4.6 +9.3 ISIS 3668490.14 +6.9 −3.3 −2.6 −7.2 ISIS 366849 0.7 +7.4 +0.1 −4.3 −2.2 ISIS 3668493.5 +8.4 −2.9 −5.2 −3.9 ISIS 366850 0.14 +11.1 −3.8 −4.6 +2.0 ISIS366850 0.7 +4.8 −0.8 −1.7 +0.9 ISIS 366850 3.5 11.2 −6.0 +4.5 +9.8

No significant change was observed in total body weight, liver weight orspleen weight at timepoints throughout or at the termination of thestudy.

Levels of BUN, creatinine, bilirubin, AST, ALT, albumin, triglycerides,cholesterol and glucose were measured in mice treated with the compoundsof the invention. Plasma samples were analyzed using the Olympus AU400eautomated chemistry analyzer (Olympus America, Irving, Tex.). Theresults, expressed as units measured, are shown in Tables 68-72. TABLE68 Effect of mixed backbone antisense compound ISIS 257016 on indicatorsof liver and kidney function Units measured per Serum Normal dose ofISIS 257016 indicator Range Saline 0.14 μM/kg 0.7 μM/kg 3.5 μM/kg BUN 15-40 31 32 32 31 mg/dL Creatinine 0.0-1.0 0.2 0.2 0.2 0.2 mg/LBilirubin 0.1-1.0 0.2 0.1 0.1 0.1 mg/dL AST  30-300 82 68 85 117 IU/LALT  30-200 22 24 26 32 IU/L Albumin 2.5-4.0 3.0 3.2 3.1 3.1 g/dLTriglycerides  25-100* 225 266 308 225 mg/dL Cholesterol  70-125 123 128128 147 mg/dL Glucose  80-150* 181 195 187 183 mg/dL

TABLE 69 Effect of mixed backbone antisense compound ISIS 366847 onindicators of liver and kidney function Units measured per Serum Normaldose of ISIS 366847 indicator Range Saline 0.14 μM/kg 0.7 μM/kg 3.5μM/kg BUN  15-40 31 29 32 29 mg/dL Creatinine 0.0-1.0 0.2 0.2 0.2 0.2mg/L Bilirubin 0.1-1.0 0.2 0.1 0 0.1 mg/dL AST  30-300 82 53 69 131 IU/LALT  30-200 22 23 28 50 IU/L Albumin 2.5-4.0 3.0 3.1 3.2 3.0 g/dLTriglycerides  25-100* 225 289 308 184 mg/dL Cholesterol  70-125 123 122132 145 mg/dL Glucose  80-150* 181 173 193 181 mg/dL

TABLE 70 Effect of mixed backbone antisense compound ISIS 366848 onindicators of liver and kidney function Units measured per dose of ISIS366848 Serum Normal 0.14 μM/ 0.7 μM/ 3.5 μM/ indicator Range Saline kgkg kg BUN  15-40 31 31 29 32 mg/dL Creatinine 0.0-1.0 0.2 0.2 0.2 0.2mg/L Bilirubin 0.1-1.0 0.2 0.1 0.1 0.1 mg/dL AST  30-300 82 82 105 123IU/L ALT  30-200 22 23 34 46 IU/L Albumin 2.5-4.0 3.0 3.1 3.1 3.0 g/dLTriglycerides  25-100* 225 320 374 246 mg/dL Cholesterol  70-125 123 132142 147 mg/dL Glucose  80-150* 181 200 187 190 mg/dL

TABLE 71 Effect of mixed backbone antisense compound ISIS 366849 onindicators of liver and kidney function Units measured per dose of ISIS366849 Serum Normal 0.14 μM/ 0.7 μM/ 3.5 μM/ indicator Range Saline kgkg kg BUN  15-40 31 25 30 33 mg/dL Creatinine 0.0-1.0 0.2 0.2 0.2 0.2mg/L Bilirubin 0.1-1.0 0.2 0.1 0.1 0.1 mg/dL AST  30-300 82 98 90 92IU/L ALT  30-200 22 26 24 33 IU/L Albumin 2.5-4.0 3.0 3.0 3.0 3.0 g/dLTriglycerides  25-100* 225 354 308 240 mg/dL Cholesterol  70-125 123 133129 150 mg/dL Glucose  80-150* 181 170 173 192 mg/dL

TABLE 72 Effect of mixed backbone antisense compound ISIS 366850 onindicators of liver and kidney function Units measured per dose of ISIS366850 Serum Normal 0.14 μM/ 0.7 μM/ 3.5 μM/ indicator Range Saline kgkg kg BUN  15-40 31 26 25 23 mg/dL Creatinine 0.0-1.0 0.2 0.2 0.2 0.2mg/L Bilirubin 0.1-1.0 0.2 0.1 0.1 0 mg/dL AST  30-300 82 83 69 108 IU/LALT  30-200 22 21 27 38 IU/L Albumin 2.5-4.0 3.0 3.0 3.0 3.0 g/dLTriglycerides  25-100* 225 320 380 271 mg/dL Cholesterol  70-125 123 127131 164 mg/dL Glucose  80-150* 181 192 187 179 mg/dL*Triglyceride and glucose levels are routinely higher in the Balb/cstrain of mice than in other strains of mice.

Some oligonucleotide treated animals exhibited elevated levels ofcholesterol; however, saline control animals also demonstratedcholelsterol levels at the high end of the normal range. Thus, theslightly elevated cholesterol levels do not appear to be significant.Otherwise, the levels of routine clinical indicators of liver and kidneyinjury and disease are within normal ranges and are not significantlychanged relative to saline-treated animals, demonstrating that thecompounds of the invention do not significantly affect renal or hepaticfunction. Triglyceride and glucose levels, while outside the normalrange as is common in the Balb/c strain, are not significantly elevatedrelative to saline-treated animals.

The results illustrated in this example demonstrate that mixed backbonecompounds of 16 to 20 nucleobases are delivered to the kidney, reduceSGLT2 expression in vivo, and that treatment with these compounds doesnot result in liver or kidney toxicity.

Example 33 Antisense Inhibition of SGLT2 in Sprague Dawley Rats

In accordance with the present invention, 7-week old Sprague Dawley rats(purchased from Charles River Labs, Wilmington, Mass.) were treated withSGLT2 mixed backbone compound ISIS 257016 (SEQ ID NO: 106) or SGLT2 fullphosphorothioate compound ISIS 145733 (SEQ ID NO: 106). Rats wereinjected i.p. twice a week for three weeks with 10 mg/kg ofoligonucleotide. Saline-injected animals served as controls. The ratswere sacrificed 2 days following administration of the final dose ofoligonucleotide or saline.

Rats were evaluated for SGLT2 levels in kidney. Target levels weredetermined by quantitative real-time PCR as described in other examplesherein. PCR results were normalized to cyclophilin. The data areexpressed as percent change relative to saline treated animals (“+”indicates an increase, “−” indicates a decrease) and are illustrated inTable 73. TABLE 73 Antisense inhibition of SGLT2 mRNA expression inSprague Dawley rats (expressed as percent change in SGLT2 mRNAexpression relative to saline) % Change Treatment in mRNA Saline 0 ISIS257016 −83.9 ISIS 145733 −38.5

These results illustrate that both full phosphorothioate and mixedbackbone compounds inhibit SGLT2 expression in the kidney of rats.However, the mixed backbone compound is a more effective inhibitor ofSGLT2.

Treated rats were further evaluated for body weight, kidney weight,liver weight and spleen weight. For body weight, the data are expressedas percent change in body weight (“+” indicates an increase, “−”indicates a decrease). For organ weights, the results are expressed aspercent of saline control normalized to body weight. The results arepresented in Table 74 and Table 75. TABLE 74 Effects of antisensecompounds on total body weight of rats (expressed as percent change inweight) Body Treatment weight Saline +60.7 ISIS 257016 +58.4 ISIS 145733+57.1

TABLE 75 Effects of antisense compounds on total kidney weight, liverweight and spleen weight of rats (expressed as percent of saline controlnormalized to body weight) Kidney Liver Spleen Treatment weight weightweight ISIS 257016 99.3 93.4 105.8 ISIS 145733 107.2 105.2 123.4

No significant change was observed in total body weight, kidney weight,liver weight or spleen weight at timepoints throughout or at thetermination of the study.

Levels of BUN, creatinine, bilirubin, AST, ALT, albumin, triglycerides,cholesterol and glucose were measured in rats treated with the compoundsof the invention. Plasma samples were analyzed using the Olympus AU400eautomated chemistry analyzer (Olympus America, Irving, Tex.). Theresults, expressed as units measured, are shown in Table 76. TABLE 76Effect of mixed backbone antisense compound ISIS 257016 and fullphosphorothioate compound ISIS 145733 on indicators of liver and kidneyfunction (expressed as units measured) ISIS ISIS Serum Indicator Saline257016 145733 BUN 19 19 17 mg/dL Creatinine 0.3 0.4 0.2 mg/L Bilirubinmg/dL 0.1 0.1 0.1 AST 157 105 105 IU/L ALT 65 44 36 IU/L Albumin 3.7 3.83.6 g/dL Triglycerides 42 47 53 mg/dL Cholesterol 68 66 54 mg/dL Glucose189 173 180 mg/dL

The levels of routine clinical indicators of liver and kidney injury arenot significantly changed relative to saline-treated animals,demonstrating that the compounds of the invention do not significantlyaffect renal or hepatic function in rats.

The results illustrated in this example demonstrate that both fullphosphorothioate and mixed backbone compounds are delivered to thekidney, reduce SGLT2 expression in vivo, and that treatment with thesecompounds does not result in liver or kidney toxicity. The resultsfurther indicate that mixed backbone compounds are more effectiveinhibitors of SGLT2 expression in vivo.

Various modifications of the invention, in addition to those describedherein, will be apparent to those skilled in the art from the foregoingdescription. Such modifications are also intended to fall within thescope of the appended claims. Each reference (including, but not limitedto, journal articles, U.S. and non-U.S. patents, patent applicationpublications, international patent application publications, gene bankaccession numbers, and the like) cited in the present application isincorporated herein by reference in its entirety.

1. An oligomeric compound 8 to 80 nucleobases in length targeted to anucleic acid molecule encoding SGLT2, wherein the compound is at least70% complementary to the nucleic acid molecule encoding SGLT2, andwherein the compound inhibits the expression of SGLT2 mRNA by at least10%.
 2. The compound of claim 1 comprising 10 to 50 nucleobases inlength.
 3. The compound of claim 2 comprising 13 to 30 nucleobases inlength.
 4. The compound of claim 3 comprising 15 to 25 nucleobases inlength.
 5. The compound of claim 4 comprising 18 to 22 nucleobases inlength.
 6. The compound of claim 1 comprising an oligonucleotide.
 7. Thecompound of claim 6 comprising a DNA oligonucleotide.
 8. The compound ofclaim 6 comprising an RNA oligonucleotide.
 9. The compound of claim 6comprising a chimeric oligonucleotide.
 10. The compound of claim 6wherein at least a portion of the compound hybridizes with RNA to forman oligonucleotide-RNA duplex.
 11. The compound of claim 1 comprising atleast 80% complementarity with the nucleic acid molecule encoding SGLT2.12. The compound of claim 1 comprising at least 90% complementarity withthe nucleic acid molecule encoding SGLT2.
 13. The compound of claim 1comprising at least 95% complementarity with the nucleic acid moleculeencoding SGLT2.
 14. The compound of claim 1 comprising at least 99%complementarity with the nucleic acid molecule encoding SGLT2.
 15. Thecompound of claim 1 comprising at least one modified internucleosidelinkage, sugar moiety, or nucleobase.
 16. The compound of claim 1comprising at least one 2′-O-methoxyethyl sugar moiety.
 17. The compoundof claim 1 comprising at least one phosphorothioate internucleosidelinkage.
 18. The compound of claim 1 wherein at least one cytosine is a5-methylcytosine.
 19. A method of inhibiting the expression of SGLT2 ina cell or tissue comprising contacting the cell or tissue with thecompound of claim 1 so that expression of SGLT2 is inhibited.
 20. Amethod of screening for a modulator of SGLT2 comprising: contacting atarget segment of a nucleic acid molecule encoding SGLT2 with one ormore candidate modulators of SGLT2; and identifying one or moremodulators of SGLT2 expression which modulate the expression of SGLT2.21. The method of claim 20 wherein the modulator of SGLT2 expressioncomprises an oligonucleotide, an antisense oligonucleotide, a DNAoligonucleotide, an RNA oligonucleotide, an RNA oligonucleotide havingat least a portion of said RNA oligonucleotide capable of hybridizingwith RNA to form an oligonucleotide-RNA duplex, or a chimericoligonucleotide.
 22. A method for identifying a disease state comprisingidentifying the presence of SGLT2 in a sample using at least one primercomprising SEQ ID NO: 5 or SEQ ID NO: 6, or a probe comprising SEQ IDNO:
 7. 23. A kit or assay device comprising the compound of claim
 1. 24.A method of treating an animal having a disease or condition associatedwith SGLT2 comprising administering to the animal a therapeutically orprophylactically effective amount of the compound of claim 1 so thatexpression of SGLT2 is inhibited.
 25. The method of claim 24 wherein thedisease or condition is a hyperproliferative or metabolic disorder. 26.The compound of claim 1 comprising at least an 8-nucleobase portion ofSEQ ID NO: 19, 20, 21, 22, 23, 26, 27, 28, 29, 32, 33, 34, 35, 37, 38,39, 40, 41, 43, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 61,62, 63, 64, 66, 67, 68, 69, 70, 73, 74, 77, 79, 80, 82, 85, 88, 90, 91,92, 93, 94, or
 95. 27. The compound of claim 26 wherein the compoundcomprises SEQ ID NO: 19, 20, 21, 22, 23, 26, 27, 28, 29, 32, 33, 34, 35,37, 38, 39, 40, 41, 43, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 61, 62, 63, 64, 66, 67, 68, 69, 70, 73, 74, 77, 79, 80, 82, 85, 88,90, 91, 92, 93, 94, or
 95. 28. The compound of claim 1 wherein thecompound comprises at least an 8-nucleobase portion of SEQ ID NO: 99,105, 107, 109, 110, 117, 121, 124, 125, 126, 130, 131, 132, 136, 138,139, 142, 147, 148, 150, 153, 155, 162, 173, or
 175. 29. The compound ofclaim 28 wherein the compound comprises SEQ ID NO: 99, 105, 107, 109,110, 117, 121, 124, 125, 126, 130, 131, 132, 136, 138, 139, 142, 147,148, 150, 153, 155, 162, 173, or
 175. 30. The compound of claim 1wherein the compound comprises an antisense nucleic acid molecule thatis specifically hybridizable with a 5′-untranslated region of a nucleicacid molecule encoding SGLT2.
 31. The compound of claim 1 wherein thecompound comprises an antisense nucleic acid molecule that isspecifically hybridizable with a start region of a nucleic acid moleculeencoding SGLT2.
 32. The compound of claim 1 wherein the compoundcomprises an antisense nucleic acid molecule that is specificallyhybridizable with a coding region of a nucleic acid molecule encodingSGLT2.
 33. The compound of claim 1 wherein the antisense compoundcomprises an antisense nucleic acid molecule that is specificallyhybridizable with a stop region of a nucleic acid molecule encodingSGLT2.
 34. The compound of claim 1 wherein the antisense compoundcomprises an antisense nucleic acid molecule that is specificallyhybridizable with a 3′-untranslated region of a nucleic acid moleculeencoding SGLT2.
 35. The compound of claim 1 which comprises a firstregion consisting of at least 5 contiguous 2′-deoxy.nucleosides flankedby a second region and a third region, wherein each of the second andthird regions, independently, comprises at least one 2′-O-methoxyethylnucleoside, and wherein the internucleoside linkages of the first regionare phosphorothioate linkages and the internucleoside linkages of thesecond and third regions are phosphodiester linkages.
 36. A method ofinhibiting the expression of SGLT2 in a kidney cell or kidney tissuecomprising contacting the kidney cell or kidney tissue with the compoundof claim
 35. 37. A method of enhancing inhibition of expression of SGLT2in a kidney cell or kidney tissue comprising contacting the kidney cellor kidney tissue with the compound of claim 35 so that expression ofSGLT2 is inhibited.
 38. The method of claim 37 wherein the compoundcomprises SEQ ID NO: 106, 255, or
 256. 39. A method of preventing ordelaying the onset of a disease or condition in an animal comprisingadministering to the animal an effective amount of the compound of claim35 so that expression of SGLT2 is inhibited, wherein the disease orcondition is associated with expression of SGLT2 in the kidney.
 40. Themethod of claim 39 wherein the compound comprises SEQ ID NO: 106, 255,or
 256. 41. A method of preventing or delaying the onset of type 2diabetes in an animal comprising administering to the animal thecompound of claim 35 so that expression of SGLT2 is inhibited.
 42. Themethod of claim 41 wherein said animal is a primate or a rodent.
 43. Themethod of claim 41 wherein the compound comprises SEQ ID NO: 106, 255,or
 256. 44. A method of preventing or delaying the onset of an increasein blood glucose level in an animal comprising administering to theanimal the compound of claim 35 so that expression of SGLT2 isinhibited.
 45. The method of claim 44 wherein the animal is a primate ora rodent.
 46. The method of claim 44 wherein the blood glucose level isplasma glucose level or serum glucose level.
 47. The method of claim 44wherein the animal is a diabetic animal.
 48. The method of claim 44wherein the animal is insulin-resistant as compared to a normal animal.49. The method of claim 44 wherein the compound comprises SEQ ID NO:106, 255, or
 256. 50. A method of decreasing blood glucose level in ananimal comprising administering to the animal the compound of claim 35so that expression of SGLT2 is inhibited.
 51. The method of claim 50wherein the animal is a primate or a rodent.
 52. The method of claim 50wherein the blood glucose level is plasma glucose level or serum glucoselevel.
 53. The method of claim 50 wherein the animal is a diabeticanimal.
 54. The method of claim 50 wherein the animal isinsulin-resistant as compared to a normal animal.
 55. The method ofclaim 50 wherein the compound comprises SEQ ID NO: 106, 255, or
 256. 56.A method of enhancing inhibition of expression of SGLT2 in a kidney cellor kidney tissue comprising contacting the cell or tissue with anantisense compound comprising a first central region comprising at least5 contiguous 2′-deoxy nucleosides flanked by a second 5′ region and athird 3′ region, wherein each of the second and third regions,independently, comprises at least one 2′-O-methoxyethyl nucleoside, andwherein the internucleoside linkages of the first region arephosphorothioate linkages and the internucleoside linkages of the secondand third regions are phosphodiester linkages except one or both of theextreme 5′ linkage and the extreme 3′ linkage are phosphorothioatelinkages, so that expression of the RNA target is inhibited.